Method for producing a purified hemoglobin product

ABSTRACT

A method for producing a purified hemoglobin product includes loading a hemoglobin solution onto an anion exchange chromatography column. At least one tris(hydroxymethyl) aminomethane acetate buffer solution is injected into the column. The buffer solution has a pH lower than that of the column, whereby a purified hemoglobin product elutes from the column. In one embodiment, the hemoglobin solution initially can be equilibrated at a pH of greater than about 8.7. In another embodiment, contaminants can be removed by equilibrating the column with at least about eleven column void volumes of buffer solution at an intermediate pH of between about 8.2 and about 8.6, to thereby form a stepped pH gradient. In still another embodiment, all buffer solutions employed are tris(hydroxymethyl) aminomethane acetate.

RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. Ser. No. 08/473,497,filed Jun. 7, 1995, now abandoned, which is a Continuation-in-Part ofU.S. Ser. No. 08/458,916, filed Jun. 2, 1995, now U.S. Pat. No.5,840,852 which is a Continuation of U.S. Ser. No. 08/409,337, filedMar. 23, 1995, now U.S. Pat. No. 5,854,209 the teachings of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

There exists a need for a blood-substitute to treat or prevent hypoxiaresulting from blood loss (e.g, from acute hemorrhage or during surgicaloperations), resulting from anemia (e.g., pernicious anemia or sicklecell anemia), or resulting from shock (e.g, volume deficiency shock,anaphylactic shock, septic shock or allergic shock). The use of bloodand blood fractions as in these capacities as a blood-substitute isfraught with disadvantages. For example, the use of whole blood often isaccompanied by the risk of transmission of hepatitis-producing virusesand AIDS-producing viruses which can complicate patient recovery orresult in patient fatalities. Additionally, the use of whole bloodrequires blood-typing and cross-matching to avoid immunohematologicalproblems and interdonor incompatibility.

Human hemoglobin, as a blood-substitute, possesses osmotic activity andthe ability to transport and transfer oxygen, but it has thedisadvantage of rapid elimination from circulation by the renal routeand through vascular walls, resulting in a very short, and therefore, atypically unsatisfactory half-life. Further, human hemoglobin is alsofrequently contaminated with toxic levels of endotoxins, bacteria and/orviruses.

Non-human hemoglobin suffers from the same deficiencies as humanhemoglobin. In addition, hemoglobin from non-human sources is alsotypically contaminated with proteins, such as antibodies, which couldcause an immune system response in the recipient.

Previously, at least four other types of blood-substitutes have beenutilized, including perfluorochemicals, synthesized hemoglobinanalogues, liposome-encapsulated hemoglobin, and chemically-modifiedhemoglobin. However, many of these blood-substitutes have typically hadshort intravascular retention times, being removed by the circulatorysystem as foreign substances or lodging in the liver, spleen, and othertissues. Also, many of these blood-substitutes have been biologicallyincompatible with living systems.

SUMMARY OF THE INVENTION

The invention relates to a method for producing a purified hemoglobinproduct.

In one embodiment, the method includes loading a hemoglobin solutiononto an anion exchange chromatography column. At least onetris(hydroxymethyl) aminomethane acetone buffer solution is injectedinto the column, the buffer solution having a pH lower than that of thecolumn, whereby a purified hemoglobin products elutes from the column.

In another embodiment, a hemoglobin solution is loaded onto an anionexchange column, the column initially being equilibrated to a pH greaterthan about 8.7. At least one buffer solution then is injected into thecolumn, the buffer solution having a pH that is lower than about 8.6,whereby the purified hemoglobin product elutes from the column.

Still another embodiment includes loading a hemoglobin solution onto ananion exchange chromatography column. At least eleven column voidvolumes of equilibrating buffer solution, having a pH in a range ofbetween about 8.2 and about 8.6, are then injected into the column. Abuffer solution having a pH lower than that of the equilibrating buffersolution then is injected into the column, whereby a purified hemoglobinproduct elutes from the column.

In yet another embodiment, a hemoglobin solution is loaded onto an anionexchange chromatography column that has been initially calibrated to apH greater than about 8.7. At least eleven column void volumes of anequilibrating buffer solution of tris(hydroxymethyl) aminomethaneacetate, having a pH in a range of between about 8.2 and about 8.6, arethen injected into the column. A buffer solution of tris(hydroxymethyl)aminomethane acetate, having a pH lower than about 8.2, then is injectedinto the column, whereby the purified hemoglobin solution elutes fromthe column.

The method of this invention advantageously achieves a hemoglobinproduct that is substantially free of even recalcitrant proteinmaterials such as carbonic anhydrase. The method can also obtain arelatively high yield of hemoglobin from a solution that includes manycontaminants. Thus, the hemoglobin derived from one species can besuccessfully used in a different species as a blood-substitute withoutthe recipient species suffering significant side effects.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the process of the invention will nowbe more particularly described with reference to the accompanyingdrawings and pointed out in the claims. It will be understood that theparticular embodiments of the invention are shown by way of illustrationand not as limitations of the invention. The principle features of thisinvention can be employed in various embodiments without departing fromthe scope of the present invention.

The invention relates to a method for producing a purified hemoglobinproduct substantially free of other blood protein components andcontaminants, employing a chromatographic column. The method ischaracterized in the use of a pH gradient to elute the hemoglobincomponent.

Concentrated Hb solution obtained from the disruption, fractionationand/or ultrafiltration of red blood cells, is directed into one or moreparallel chromatographic columns to further separate the hemoglobin byhigh performance liquid chromatography from other contaminants such asantibodies, endotoxins, phospholipids, enzymes (such as, carbonicanhydrase), viruses and transmissible spongiform encephalopathy agents.The chromatographic column contains an anion exchange medium suitable toseparate Hb from non-hemoglobin proteins. Suitable anion exchange mediainclude, for example, silica, alumina, titania gel, cross-linkeddextran, agarose or a derivatized moiety, such as a polyacrylamide, apolyhydroxyethyl-methacrylate or a styrene divinylbenzene, that has beenderivatized with a cationic chemical functionality, such as adiethylaminoethyl or quaternary aminoethyl group. A suitable anionexchange medium and corresponding eluants for the selective absorptionand desorption of Hb as compared to other proteins and contaminants,which are likely to be in a lysed RBC phase, are readily determinable byone of reasonable skill in the art.

In a more preferred embodiment, a method is used to form an anionexchange medium from silica gel which is hydrothermally treated toincrease the pore size, exposed to y-glycidoxy propylsilane to formactive epoxide groups and then exposed to C₃ H₇ (CH₃)₂ NCl to form aquaternary ammonium anion exchange medium. This method is described inthe Journal of Chromatography, 120:321-333 (1976), which is incorporatedherein by reference in its entirety. In one embodiment, thechromatographic column, or columns, are first equilibrated to a pHgreater than about 8.7. Preferably, the chromatography column isequilibrated to a pH in a range of between about 8.7 and about 10.0. Ina particularly preferred embodiment, the pH of equilibration is in arange of between about 8.7 and about 9.3 and, most preferably, in arange of between about 8.9 and about 9.1. Preferably, the bufferemployed to initially equilibrate the column is tris(hydroxymethyl)aminomethane acetate (Tris-acetate), and has a concentration of about 20mmoles/liter (mM/l).

Hemoglobin solution that, preferably, has been dialyzed against purifiedwater (U.S.P.) and, also preferably, has a conductivity of about 280μS/cm and a pH between about 6.75 and about 7.75, is then injected ontothe medium in the column to thereby load the column with hemoglobin. Theconcentration of hemoglobin solution that is loaded onto the columntypically has a concentration in a range of between about 90 and about200 grams/liter. Preferably, the concentration of the hemoglobinsolution is in a range of between about 90 and about 110 grams/liter.Preferably, after injecting the concentrated Hb solution, thechromatographic column is washed for about ten minutes (4 column voidvolumes) with the Tris-acetate to elute non-hemoglobin components thatdo not bind to the media, and to facilitate strong bonding of hemoglobinto the media.

A pH gradient is used in the chromatographic column to separate proteincontaminants, such as the enzyme carbonic anhydrase, phospholipids,antibodies and endotoxins, from the Hb. The pH gradient can be acontinuous gradient or a stepped gradient. Buffer solutions havingdifferent pH values are sequentially injected into the column to createa pH gradient of the eluate over time. It is preferred that the buffersbe filtered prior to injection, such as with a suitable 10,000 Daltondepyrogenation membrane. The buffer solutions should be of monovalentbuffers which have a low ionic strength so that elution of Hb andnon-hemoglobin contaminants is generally dependent upon pH and notsignificantly dependent upon ionic strength. Typically, buffers with anionic concentration of about 50 mM, or less, have suitably low ionicstrengths.

Preferably, the contaminants and hemoglobin of the hemoglobin solutionare separated by elution from the column in a stepped gradient, wherebya buffer is employed that has a pH between that at which the hemoglobinis loaded onto the column and a final pH, at which hemoglobin is elutedfrom the column. In one embodiment, the stepped gradient includesinjecting a suitable buffer solution into the column. The buffersolution has a pH lower than the pH of the column at the time the columninitially was loaded with hemoglobin. The column is equilibrated withthe buffer solution. Preferably, at least about six column volumes ofthe buffer solution are injected into the column. A "column volume," asdefined herein, is the volume of the column, not including any packingmaterial. Typically, suitable column packings, i.e. exchange media, foruse with the present invention, cause the column to have a void fractionof about 0.525 of the total volume of the column. Six column volumes,therefore, is equivalent to about 11.7 "column void volumes." Generally,at least about 11 column void volumes of buffer solution are injectedinto the column.

Preferably, the pH of the buffer solution is lower than about 8.6. Morepreferably, the pH of the buffer solution is in a range of between about8.2 and about 8.4. In an especially preferred embodiment, the buffersolution is tris(hydroxymethyl) aminomethane acetate (Tris-acetate).Contaminants of the hemoglobin solution are eluted from the column byequilibrating the column in this manner.

Thereafter, a buffer is injected into the column to elute hemoglobin.Preferably, the buffer solution is of tris(hydroxymethyl) aminomethaneacetate. More preferably, the Tris-acetate solution has a pH in a rangeof between about 6.5 and about 7.5. The hemoglobin eluate is thepurified hemoglobin product.

In a preferred embodiment, the first 3%-to-4% of the Hb eluate and thelast 3%-to-4% of the Hb eluate are directed to waste to provideassurance of the purity of the Hb eluate. It is preferred that the Hbeluate be directed through a sterile filter. Suitable sterile filtersinclude 0.22 μm filters, such as a Sartorius Sartobran Cat # 5232507G1PH filter.

Wherein the chromatographic columns are to be reused, contaminatingnon-hemoglobin proteins and endotoxin remaining in the columns are theneluted by a fourth buffer. An example of a suitable buffer solution is aNaCl/Tris-acetate solution (concentrations about 1.0 M NaCl and about 20mM Tris-acetate; pH about 8.4 to about 9.4, preferably about 8.9-9.1).In a most preferred embodiment, all of the buffer solutions are oftris(hydroxymethyl) aminomethane acetate. Typically, the buffersolutions used are at a temperature in a range of between about 0° C.and about 50° C. Preferably, buffer temperature is about 12.4±1.0° C.during use. In addition, the buffers are typically stored at atemperature in a range of between about 9° C. and about 11° C.

As defined herein, a blood-substitute is a hemoglobin-based oxygencarrying composition for use in humans, mammals and other vertebrates,which is capable of transporting and transferring oxygen to vital organsand tissues, at least, and can maintain sufficient intravascular oncoticpressure. A vertebrate is as classically defined, including humans, orany other vertebrate animals which uses blood in a circulatory system totransfer oxygen to tissue. Additionally, the definition of circulatorysystem is as classically defined, consisting of the heart, arteries,veins and microcirculation including smaller vascular structures such ascapillaries.

A blood-substitute formed by the method of invention preferably is madeaccording to one embodiment of the invention must have levels ofendotoxins, phospholipids, foreign proteins and other contaminants whichwill not result in a significant immune system response and which arenon-toxic to the recipient. Preferably, a blood-substitute substitute isultrapure. "Ultrapure," as defined herein, means containing less than0.5 EU/ml of endotoxin, less than 3.3 nmoles/ml phospholipids and littleto no detectable levels of non-hemoglobin proteins, such as serumalbumin or antibodies.

The term "endotoxin" refers to the cell-bound lipopolysaccharides,produced as a part of the outer layer of gram-negative bacterial cellwalls, which under many conditions are toxic. When injected intoanimals, endotoxins can cause fever, diarrhea, hemorrhagic shock, andother tissue damage. Endotoxin unit (EU) has been defined by the UnitedStates Pharmacopeial Convention of 1983, page 3014, as the activitycontained in 0.1 nanograms of U.S. reference standard lot EC-5. One vialof EC-5 contains 10,000 EU. Examples of suitable means for determiningendotoxin concentrations in a blood-substitute include the method"Kinetic/ Turbidimetric Limuus Amebocytic Lystate (LAL) 5000Methodology" developed by Associates of Cape Cod, Woods Hole, Mass.

"Stable polymerized hemoglobin," as defined herein, is ahemoglobin-based oxygen carrying composition which does notsubstantially increase or decrease in molecular weight distributionand/or in methemoglobin content during storage periods at suitablestorage temperatures for periods of two years or more, and preferablyfor periods of two years or more, when stored in a low oxygenenvironment. Suitable storage temperatures for storage of one year ormore are between about 0° C. and about 40° C. The preferred storagetemperature range is between about 0° C. and about 25° C.

A suitable low oxygen environment, or an environment that issubstantially oxygen-free, is defined as the cumulative amount of oxygenin contact with the blood-substitute, over a storage period of at leastabout two months, preferably at least about one year, or more preferablyat least about two years which will result in a methemoglobinconcentration of less than about 15% by weight in the blood-substitute.The cumulative amount of oxygen includes oxygen inleakage into theblood-substitute packaging and the original oxygen content of theblood-substitute and packaging.

Throughout this method, from red blood cell (RBC) collection untilhemoglobin polymerization, blood solution, RBCs and hemoglobin aremaintained under conditions sufficient to minimize microbial growth, orbioburden, such as maintaining temperature at less than about 20° C. andabove 0° C. Preferably, temperature is maintained at a temperature ofabout 15° C. or less. More preferably, the temperature is maintained at10±2° C.

In this method, portions of the components for the process for preparinga stable polymerized hemoglobin blood-substitute are sufficientlysanitized to produce a sterile product. Sterile is as defined in theart, specifically, that the solution meets United States Pharmacopeiarequirements for sterility provided in USP XXII, Section 71, pages1483-1488. Further, portions of components that are exposed to theprocess stream, are usually fabricated or clad with a material that willnot react with or contaminate the process stream. Such materials caninclude stainless steel and other steel alloys, such as Inconel.

Suitable RBC sources include human blood, bovine blood, ovine blood,porcine blood, blood from other vertebrates and transgenically-producedhemoglobin, such as the transgenic Hb described in BIO/TECHNOLOGY, 12:55-59 (1994).

The blood can be collected from live or freshly slaughtered donors. Onemethod for collecting bovine whole blood is described in U.S. Pat. Nos.5,084,558 and 5,296,465, issued to Rausch et al. It is preferred thatthe blood be collected in a sanitary manner.

At or soon after collection, the blood is mixed with at least oneanticoagulant to prevent significant clotting of the blood. Suitableanticoagulants for blood are as classically known in the art andinclude, for example, sodium citrate, ethylenediaminetetraacetic acidand heparin. When mixed with blood, the anticoagulant may be in a solidform, such as a powder, or in an aqueous solution.

It is understood that the blood solution source can be from a freshlycollected sample or from an old sample, such as expired human blood froma blood bank. Further, the blood solution could previously have beenmaintained in frozen and/or liquid state. It is preferred that the bloodsolution is not frozen prior to use in this method.

In another embodiment, prior to introducing the blood solution toanticoagulants, antibiotic levels in the blood solution, such aspenicillin, are assayed. Antibiotic levels are determined to provide adegree of assurance that the blood sample is not burdened with aninfecting organism by verifying that the donor of the blood sample wasnot being treated with an antibiotic. Examples of suitable assays forantibiotics include a penicillin assay kit (Difco, Detroit, Mich.)employing a method entitled "Rapid Detection of Penicillin in Milk". Itis preferred that blood solutions contain a penicillin level of lessthan or equal to about 0.008 units/ml. Alternatively, a herd managementprogram to monitor the lack of disease in or antibiotic treatment of thecattle may be used.

Preferably, the blood solution is strained prior to or during theanticoagulation step, for example by straining, to remove largeaggregates and particles. A 600 mesh screen is an example of a suitablestrainer.

The RBCs in the blood solution are then washed by suitable means, suchas by diafiltration or by a combination of discrete dilution andconcentration steps with at least one solution, such as an isotonicsolution, to separate RBCs from extracellular plasma proteins, such asserum albumins or antibodies (e.g., immunoglobulins (IgG)). It isunderstood that the RBCs can be washed in a batch or continuous feedmode.

Acceptable isotonic solutions are as known in the art and includesolutions, such as a citrate/saline solution, having a pH and osmolaritywhich does not rupture the cell membranes of RBCs and which displacesplasma portion of the whole blood. A preferred isotonic solution has aneutral pH and an osmolarity between about 285-315 mOsm. In a preferredembodiment, the isotonic solution is composed of an aqueous solution ofsodium citrate dihydrate (6.0 g/l) and of sodium chloride (8.0 g/l).

Water which can be used in the method of invention include distilledwater, deionized water, water-for-injection (WFI) and/or low pyrogenwater (LPW). WFI, which is preferred, is deionized, distilled water thatmeets U.S. Pharmacological Specifications for water-for-injection. WFIis further described in Pharmaceutical Engineering, 11, 15-23 (1991).LPW, which is preferred, is deionized water containing less than 0.002EU/ml.

It is preferred that the isotonic solution be filtered prior to beingadded to the blood solution. Examples of suitable filters include aMillipore 10,000 Dalton ultrafiltration membrane, such as a MilliporeCat # CDUF 050 G1 filter or A/G Technology hollow fiber, 10,000 Dalton(Cat # UFP-10-C-85).

In a preferred embodiment, RBCs in the blood solution are washed bydiafiltration. Suitable diafilters include microporous membranes withpore sizes which will separate RBCs from substantially smaller bloodsolution components, such as a 0.1 μm to 0.5 μm filter (e.g., a 0.2 μmhollow fiber filter, Microgon Krosflo II microfiltration cartridge).Concurrently, filtered isotonic solution is added continuously (or inbatches) as makeup at a rate equal to the rate (or volume) of filtratelost across the diafilter. During RBC washing, components of the bloodsolution which are significantly smaller in diameter than RBCs, or arefluids such as plasma, pass through the walls of the diafilter in thefiltrate. RBCs, platelets and larger bodies of the diluted bloodsolution, such as white blood cells, are retained and mixed withisotonic solution, which is added continuously or batchwise to form adialyzed blood solution.

In a more preferred embodiment, the volume of blood solution in thediafiltration tank is initially diluted by the addition of a volume of afiltered isotonic solution to the diafiltration tank. Preferably, thevolume of isotonic solution added is about equal to the initial volumeof the blood solution.

In an alternate embodiment, the RBCs are washed through a series ofsequential (or reverse sequential) dilution and concentration steps,wherein the blood solution is diluted by adding at least one isotonicsolution, and is concentrated by flowing across a filter, therebyforming a dialyzed blood solution.

RBC washing is complete when the level of plasma proteins contaminatingthe RBCs has been substantially reduced (typically at least about 90%).Typically, RBC washing is complete when the volume of filtrate drainedfrom diafilter 34 equals about 300%, or more, of the volume of bloodsolution contained in the diafiltration tank prior to diluting the bloodsolution with filtered isotonic solution. Additional RBC washing mayfurther separate extracellular plasma proteins from the RBCs. Forinstance, diafiltration with 6 volumes of isotonic solution may removeat least about 99% of IgG from the blood solution.

The dialyzed blood solution is then exposed to means for separating theRBCs in the dialyzed blood solution from the white blood cells andplatelets, such as by centrifugation.

It is understood that other methods generally known in the art forseparating RBCs from other blood components can be employed. Forexample, sedimentation, wherein the separation method does not rupturethe cell membranes of a significant amount of the RBCs, such as lessthan about 30% of the RBCs, prior to RBC separation from the other bloodcomponents.

Following separation of the RBCs, the RBCs are lysed by a means forlysing RBCs to release hemoglobin from the RBCs to form ahemoglobin-containing solution. Lysis means can use various lysismethods, such as mechanical lysis, chemical lysis, hypotonic lysis orother known lysis methods which release hemoglobin without significantlydamaging the ability of the Hb to transport and release oxygen.

In yet another embodiment, recombinantly produced hemoglobin, such asthe recombinantly produced hemoglobin described in Nature, 356: 258-260(1992), can be processed in the method of invention in place of RBCs.The bacteria cells containing the hemoglobin are washed and separatedfrom contaminants as described above. These bacteria cells are thenmechanically ruptured by means known in the art, such as a ball mill, torelease hemoglobin from the cells and to form a lysed cell phase. Thislysed cell phase is then processed as is the lysed RBC phase.

Following lysis, the lysed RBC phase is then ultrafiltered to removelarger cell debris, such as proteins with a molecular weight above about100,000 Daltons. Generally, cell debris include all whole and fragmentedcellular components with the exception of Hb, smaller cell proteins,electrolytes, coenzymes and organic metabolic intermediates. Acceptableultrafilters include, for example, 100,000 Dalton filters made byMillipore (Cat # CDUF 050 H1) and made by A/G Technology (Needham,Mass.; Model No. UFP100E55).

It is preferred that ultrafiltration continues until the concentrationof Hb in the lysed RBC phase is less than 8 grams/liter (g/l) tomaximize the yield of hemoglobin available for polymerization. Othermethods for separating Hb from the lysed RBC phase can be employed,including sedimentation, centrifugation or microfiltration.

The Hb ultrafiltrate can then be ultrafiltered to remove smaller celldebris, such as electrolytes, coenzymes, metabolic intermediates andproteins less than about 30,000 Daltons in molecular weight, and waterfrom the Hb ultrafiltrate. Suitable ultrafilters include a 30,000 Daltonultrafilter (Millipore Cat # CDUF 050 T1 and/or Armicon, # 540 430).

The concentrated Hb solution can then be directed into one or moreparallel chromatographic columns as described in more detail above.

The Hb eluate obtained from the chromatography step is then preferablydeoxygenated prior to polymerization to form a deoxygenated Hb solution(hereinafter deoxy-Hb)by means that substantially deoxygenate the Hbwithout significantly reducing the ability of the Hb in the Hb eluate totransport and release oxygen, such as would occur from denaturation orformation of oxidized hemoglobin (met Hb).

In one embodiment, the Hb eluate is deoxygenated by gas transfer of aninert gas across a phase membrane. Such inert gases include, forexample, nitrogen, argon and helium. It is understood that other meansfor deoxygenating a solution of hemoglobin, which are known in the art,can be used to deoxygenate the Hb eluate. Such other means, can include,for example, nitrogen sparging of the Hb eluate, chemical scavengingwith reducing agents such as N-acetyl-L-cysteine (NAC), cysteine, sodiumdithionite or ascorbate, or photolysis by light.

Following elution from the chromatographic column, the Hb eluate ispreferably concentrated to improve the efficiency of the process. The Hbeluate is recirculated through an ultrafilter to concentrate the Hbeluate to form a concentrated Hb solution. Suitable ultrafiltersinclude, for example, 30,000 or less Dalton ultrafilters (e.g.,Millipore Helicon, Cat # CDUF050G1 or Amicon Cat # 540430). Typically,concentration of the Hb eluate is complete when the concentration of Hbis between about 100 to about 120 g/l. While concentrating the Hbeluate, the Hb eluate temperature is preferably maintained atapproximately 8-12° C.

Buffer is then directed into the Hb solution, which is preferablyconcentrated, to adjust the ionic strength of the Hb solution to enhanceHb deoxygenation. It is preferred that the ionic strength be adjusted tobetween about 150 meq/l and about 200 meq/l to reduce the oxygenaffinity of the Hb in the Hb solution. Suitable buffers include bufferswith a pH that will not result in significant denaturing of the Hbprotein but will have an ionic strength sufficiently high to promote Hbdeoxygenation. Examples of suitable buffers include saline solutionswith a pH range of about 6.5 to about 8.9. A preferred buffer is anaqueous 1.0 M NaCl, 20 mM Tris-acetate solution with a pH of about 8.9.

Preferably, the resulting buffered Hb solution is then recirculatedthrough the ultrafilter, to again concentrate the Hb solution to improvethe efficiency of the process. In a preferred embodiment, concentrationis complete when the concentration of Hb is about 100 g/l to about 120g/l.

During deoxygenation the Hb solution is circulated through a suitablephase transfer membrane. Appropriate phase transfer membranes include,for example, a 0.05 μm polypropylene hollow fiber microfilter (e.g.,Hoechst-Celanese Cat # 5PCM-107). Concurrently, a counterflow of aninert gas is passed across the phase transfer membrane. Suitable inertgases include, for example, nitrogen, argon and helium. Gas exchangeacross phase transfer membrane thereby strips oxygen out of the Hbsolution.

Deoxygenation continues until the pO₂ of the Hb solution is reduced to alevel wherein the oxygenated Hb (oxyhemoglobin or HbO₂) content in theHb solution is about 20% or less. In a preferred embodiment, the HbO₂content in the Hb solution is about 10% or less.

During deoxygenation, the temperature of the Hb solution is typicallymaintained at a level that will balance the rate of deoxygenationagainst the rate of methemoglobin formation. Temperature is maintainedto limit methemoglobin content to less than 20%. An optimum temperaturewill result in less than about 5% methemoglobin content, and preferablyless than about 2.5% methemoglobin content, while still deoxygenatingthe Hb solution. Typically, during deoxygenation the temperature of theHb solution is maintained between about 19° C. and about 31° C.

During deoxygenation, and subsequently throughout the remaining steps ofthe method of invention, the Hb is maintained in a low oxygenenvironment to minimize oxygen absorption by the Hb and to maintain anHbO₂ content of less than about 20%, preferably less than about 10%.

The deoxygenated-Hb is then preferably equilibrated with a low oxygencontent storage buffer, containing a sulfhydryl compound, to form anoxidation-stabilized deoxy-Hb. Suitable sulfhydryl compounds includenon-toxic reducing agents, such as N-acetyl-L-cysteine (NAC)D,L-cysteine, y-γglutamyl-cysteine, glutathione,2,3-dimercapto-1-propanol, 1,4-butanedithiol, thioglycolate, and otherbiologically compatible sulfhydryl compounds. The oxygen content of alow oxygen content storage buffer must be low enough not tosignificantly reduce the concentration of sulfhydryl compound in thebuffer and to limit oxyhemoglobin content in oxidation stabilizeddeoxy-Hb to about 20% or less, preferably less than about 10%.Typically, the storage buffer has a pO₂ of less than about 50 torr.

In a preferred embodiment, the storage buffer should have a pH suitableto balance Hb polymerization and methemoglobin formation, typicallybetween about 7.6 and about 7.9.

The amount of a sulfhydryl compound mixed with the deoxy-Hb is an amounthigh enough to increase intramolecular cross-linking of Hb duringpolymerization and low enough not to significantly decreaseintermolecular cross-linking of Hb molecules, due to a high ionicstrength. Typically, about one mole of sulfhydryl functional groups(--SH) are needed to oxidation stabilize between about 0.25 moles toabout 5 moles of deoxy-Hb.

In a preferred embodiment, the storage buffer contains approximately25-35 mM sodium phosphate buffer (pH 7.7-7.8) and contains an amount ofNAC such that the concentration of NAC in oxidation stabilized deoxy-Hbis between about 0.003% and about 0.3%, by weight. More preferably, theNAC concentration in the oxidation stabilized deoxy-Hb is between about0.05% and about 0.2% by weight.

Preferably, the storage buffer is filtered prior to mixing with thedeoxy-Hb, such as through a 10,000 Dalton ultrafiltration membrane(Millipore Helicon Cat # CDUF050G1 or A/G Technology Maxcell Cat #UFP-10-C-75).

In one embodiment, the oxidation-stabilized deoxy-Hb then flows throughan optional filter. Suitable filters include a 0.2 μm polypropyleneprefilter and a 0.5 μm sterile microfilter (Pall Profile II, Cat #ABIY005Z7 or Gelman Supor). The deoxy-Hb is maintained under asubstantially oxygen-free atmosphere. This can be accomplished, forexample, by purging and blanketing the process apparatus with an inertgas, such as nitrogen, prior to and after filling withoxidation-stabilized deoxy-Hb.

Optionally, prior to transferring the oxidation-stabilized deoxy-Hb topolymerization, an appropriate amount of water is added to thepolymerization reactor. In one embodiment an appropriate amount of wateris that amount which would result in a solution with a concentration ofabout 10 to about 100 g/l Hb when the oxidation-stabilized deoxy-Hb isadded to the polymerization reactor. Preferably, the water isoxygen-depleted.

After the pO₂ of the water in the polymerization step is reduced to alevel sufficient to limit HbO₂ content to about 20%, typically less thanabout 50 torr, the polymerization reactor is blanketed with an inertgas, such as nitrogen. The oxidation-stabilized deoxy-Hb is thentransferred into the polymerization reactor, which is concurrentlyblanketed with an appropriate flow of an inert gas.

The temperature of the oxidation-stabilized deoxy-Hb solution inpolymerization reactor is raised to a temperature to optimizepolymerization of the oxidation-stabilized deoxy-Hb when contacted witha cross-linking agent. Typically, the temperature of theoxidation-stabilized deoxy-Hb is about 25° C. to about 45° C., andpreferably about 41° C. to about 43° C. throughout polymerization. Anexample of an acceptable heat transfer means for heating thepolymerization reactor is a jacketed heating system which is heated bydirecting hot ethylene glycol through the jacket.

The oxidation-stabilized deoxy-Hb is then exposed to a suitablecross-linking agent at a temperature sufficient to polymerize theoxidation-stabilized deoxy-Hb to form a solution of polymerizedhemoglobin (poly(Hb)) over a period of about 2 hours to about 6 hours.

Examples of suitable cross-linking agents include polyfunctional agentsthat will cross-link Hb proteins, such as glutaraldehyde,succindialdehyde, activated forms of polyoxyethylene and dextran,α-hydroxy aldehydes, such as glycolaldehyde,N-maleimido-6-aminocaproyl-(2'-nitro,4'-sulfonic acid)-phenyl ester,m-maleimidobenzoic acid-N-hydroxysuccinimide ester, succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester,m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate,sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl4-(p-maleimidophenyl)butyrate,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,N,N'-phenylene dimaleimide, and compounds belonging to the bis-imidateclass, the acyl diazide class or the aryl dihalide class, among others.

A suitable amount of a cross-linking agent is that amount which willpermit intramolecular cross-linking to stabilize the Hb and alsointermolecular cross-linking to form polymers of Hb, to thereby increaseintravascular retention. Typically, a suitable amount of a cross-linkingagent is that amount wherein the molar ratio of cross-linking agent toHb is in excess of about 2:1. Preferably, the molar ratio ofcross-linking agent to Hb is between about 20:1 to 40:1.

Preferably, the polymerization is performed in a buffer with a pHbetween about 7.6 to about 7.9, having a chloride concentration lessthan or equal to about 35 mmolar.

In a preferred embodiment, a suitable amount of the cross-linking agentis added to the oxidation-stabilized deoxy-Hb which are then mixed by ameans for mixing with low shear. A suitable low-shear mixing meansincludes a static mixer. A suitable static mixer is, for example, a"Kenics" static mixer obtained from Chemineer, Inc.

In one embodiment, recirculating the oxidation-stabilized deoxy-Hb andthe cross-linking agent through the static mixer causes turbulent flowconditions with generally uniform mixing of the cross-linking agent withthe oxidation-stabilized deoxy-Hb thereby reducing the potential forforming pockets of deoxy-Hb containing high concentrations of thecross-linking agent. Generally uniform mixing of the cross-linking agentand the deoxy-Hb reduces the formation of high molecular weight Hbpolymers, i.e. polymers weighing more than 500,000 Daltons, and alsopermits faster mixing of the cross-linking agent and the deoxy-Hb duringpolymerization. Furthermore, significant Hb intramolecular cross-linkingwill result during Hb polymerization due to the presence of a sulfhydrylcompound, preferably NAC. While the exact mechanism of the interactionof the sulfhydryl compound with glutaraldehyde and/or Hb is not known,it is presumed that the sulfhydryl compound affects Hb/cross-linkingagent chemical bonding in a manner that at least partially inhibits theformation of high molecular weight Hb polymers and preferentially formsstabilized tetrameric Hb.

Poly(Hb) is defined as having significant intramolecular cross-linkingif a substantial portion (e.g., at least about 50%) of the Hb moleculesare chemically bound in the poly(Hb), and only a small amount, such asless than about 15% are contained within high molecular weightpolymerized hemoglobin chains. High molecular weight poly(Hb) moleculesare molecules, for example, with a molecular weight above about 500,000Daltons.

In a preferred embodiment, glutaraldehyde is used as the cross-linkingagent. Typically, about 10 to about 70 grams of glutaraldehyde are usedper kilogram of oxidation-stabilized deoxy-Hb. More preferably,glutaraldehyde is added over a period of five hours until approximately29-31 grams of glutaraldehyde are added for each kilogram ofoxidation-stabilized deoxy-Hb.

After polymerization, the temperature of the poly(Hb) solution inpolymerization reactor is typically reduced to about 15° C. to about 25°C.

Wherein the cross-linking agent used is not an aldehyde, the poly(Hb)formed is generally a stable poly(Hb). Wherein the cross-linking agentused is an aldehyde, the poly(Hb) formed is generally not stable untilmixed with a suitable reducing agent to reduce less stable bonds in thepoly(Hb) to form more stable bonds. Examples of suitable reducing agentsinclude sodium borohydride, sodium cyanoborohydride, sodium dithionite,trimethylamine, t-butylamine, morpholine borane and pyridine borane.Prior to adding the reducing agent, the poly(Hb) solution is optionallyconcentrated by ultrafiltration until the concentration of the poly(Hb)solution is increased to between about 75 and about 85 g/l. An exampleof a suitable ultrafilter is a 30,000 Dalton filter (e.g., MilliporeHelicon, Cat # CDUF050LT and Amicon, Cat # 540430).

The pH of the poly(Hb) solution is then adjusted to the alkaline pHrange to preserve the reducing agent and to prevent hydrogen gasformation, which can denature Hb during the subsequent reduction.

In one embodiment, the pH is adjusted to greater than 10. The pH can beadjusted by adding a buffer solution to the poly(Hb) solution during orafter polymerization. The poly(Hb) is typically purified to removenon-polymerized hemoglobin. This can be accomplished by dialfiltrationor hydroxyapatite chromatography (see, e.g. copending U.S. Pat. No.5,691,453, issued on Nov. 25, 1997, the teachings of which areincorporated herein by reference).

Following pH adjustment, at least one reducing agent, preferably asodium borohydride solution, is added to the polymerization steptypically through the deoxygenation loop. Typically, about 5 to about 18moles of reducing agent are added per mole of Hb tetramer (per 64,000Daltons of Hb) within the poly(Hb). In a preferred embodiment, for everynine liters of poly(Hb) solution in polymerization subsystem 98, oneliter of 0.25 M sodium borohydride solution is added at a rate of 0.1 to0.12 lpm.

The pH and electrolytes of the stable poly(Hb) can then be restored tophysiologic levels to form a stable polymerized hemoglobinblood-substitute, by diafiltering the stable poly(Hb) with adiafiltration solution having a suitable pH and physiologic electrolytelevels. Preferably, the diafiltration solution is a buffer solution.

Wherein the poly(Hb) was reduced by a reducing agent, the diafiltrationsolution has an acidic pH, preferably between about 4 to about 6.

A non-toxic sulfhydryl compound can also be added to the stable poly(Hb)solution as an oxygen scavenger to enhance the stability of the finalpolymerized hemoglobin blood-substitute. The sulfhydryl compound can beadded as part of the diafiltration solution and/or can be addedseparately. An amount of sulfhydryl compound is added to establish asulfhydryl concentration which will scavenge oxygen to maintainmethemoglobin content less than about 15% over the storage period.Preferably, the sulfhydryl compound is NAC. Typically, the amount ofsulfhydryl compound added is an amount sufficient to establish asulfhydryl concentration between about 0.05% and about 0.2% by weight.

In a preferred embodiment, the blood-substitute is packaged underaseptic handling conditions while maintaining pressure with an inert,substantially oxygen-free atmosphere, in the polymerization reactor andremaining transport apparatus.

The specifications for a suitable stable polymerized hemoglobinblood-substitute formed by the method of invention are provided in TableI.

                  TABLE I                                                         ______________________________________                                        PARAMETER             RESULTS                                                 ______________________________________                                        pH (18-22° C.) Physiologically                                            acceptable                                                                   Endotoxin Physiologically                                                      acceptable                                                                   Sterility Test Meets Test                                                     Phospholipids.sup.a Physiologically                                            acceptable                                                                   Total Hemoglobin 10-250 g/l                                                   Methemoglobin <15%                                                            Oxyhemoglobin ≦10%                                                     Sodium, Na.sup.+ Physiologically                                               acceptable                                                                   Potassium, K.sup.+                                                            Chloride, Cl.sup.-                                                            Calcium, Ca.sup.++                                                            Boron                                                                         Glutaraldehyde Physiologically                                                 acceptable                                                                   N-acetyl-L-cysteine Physiologically                                            Acceptable                                                                   M.W. > 500,000 ≦15%                                                    M.W. ≦ 65,000 ≦10%                                              M.W. ≦ 32,000 ≦5%                                               Particulate Content ≧ 10μ <12/ml                                    Particulate Content ≧ 25μ <2/ml                                   ______________________________________                                         .sup.a measured in Hb before polymerization                              

The stable blood-substitute is then stored in a short-term storagecontainer or into sterile storage containers, each having a low oxygenenvironment as described in detail above. The storage container shouldalso be sufficiently impermeable to water vapor passage to preventsignificant concentration of the blood-substitute by evaporation overthe storage period. Significant concentration of the blood-substitute isconcentration resulting in one or more parameters of theblood-substitute being high out of specification.

The synthesis of a stable polymerized hemoglobin blood-substitute,formed according to the method of invention, is further described inU.S. Pat. No. 5,296,465.

Vertebrates which can receive the blood-substitute, formed by themethods of the invention include mammals, such as a human, non-humanprimate, a dog, a cat, a rat, a horse or a sheep. Further, vertebrates,which can receive said blood-substitute, includes fetuses (prenatalvertebrate), post-natal vertebrates, or vertebrates at time of birth.

A blood-substitute of the present invention can be administered into thecirculatory system by injecting the blood-substitute directly and/orindirectly into the circulatory system of the vertebrate, by one or moreinjection methods. Examples of direct injection methods includeintravascular injections, such as intravenous and intra-arterialinjections, and intracardiac injections. Examples of indirect injectionmethods include intraperitoneal injections, subcutaneous injections,such that the blood-substitute will be transported by the lymph systeminto the circulatory system, injections into the bone marrow by means ofa trocar or catheter. Preferably, the blood-substitute is administeredintravenously.

The vertebrate being treated can be normovolemic, hypervolemic orhypovolemic prior to, during, and/or after infusion of theblood-substitute. The blood-substitute can be directed into thecirculatory system by methods such as top loading and by exchangemethods.

A blood-substitute can be administered therapeutically, to treat hypoxictissue within a vertebrate resulting from many different causesincluding reduced RBC flow in a portion of, or throughout, thecirculatory system, anemia and shock. Further, the blood-substitute canbe administered prophylactically to prevent oxygen-depletion of tissuewithin a vertebrate, which could result from a possible or expectedreduction in RBC flow to a tissue or throughout the circulatory systemof the vertebrate. Further discussion of the administration ofhemoglobin to therapeutically or prophylactically treat hypoxia,particularly from a partial arterial obstruction or from a partialblockage in microcirculation, and the dosages used therein, is providedcopending U.S. patent application Ser. No. 08/409,337, filed Mar. 23,1995, which is incorporated herein by reference in its entirety.

Typically, a suitable dose, or combination of doses of blood-substitute,is an amount which when contained within the blood plasma will result ina total hemoglobin concentration in the vertebrate's blood between about0.1 to about 10 grams Hb/d1, or more, if required to make up for largevolume blood losses.

The invention will now be further and specifically described by thefollowing examples.

EXAMPLE 1 Synthesis of Stable Polymerized Hb Blood-Substitute

As described in U.S. Pat. No. 5,296,465, samples of bovine whole bloodwere collected, mixed with a sodium citrate anticoagulant to form ablood solution, and then analyzed for endotoxin levels.

Each blood solution sample was maintained after collection at atemperature of about 2° C. and then strained to remove large aggregatesand particles with a 600 mesh screen.

Prior to pooling, the penicillin level in each blood solution sample wasassayed with an assay kit purchased from Difco, Detroit, Mich. using themethod entitled "Rapid Detection of Penicillin in Milk" to ensure thatpenicillin levels in the blood solutions were ≦0.008 units/ml.

The blood solution samples were then pooled and mixed with depyrogenatedaqueous sodium citrate solution to form a 0.2% by weight solution ofsodium citrate in bovine whole blood (hereafter "0.2% sodium citrateblood solution").

The 0.2% sodium citrate blood solution was then passed, in-series,through 800 μm and 50 μm polypropylene filters to remove large bloodsolution debris of a diameter approximately 50 μm or more.

The RBCs were then washed to separate extracellular plasma proteins,such as BSA or IgG, from the RBCs. To wash the RBCs contained in theblood solution, the volume of blood solution in the diafiltration tankwas initially diluted by the addition of an equal volume of a filteredisotonic solution to diafiltration tank. The isotonic solution wasfiltered with a Millipore (Cat # CDUF 050 G1) 10,000 Daltonultrafiltration membrane. The isotonic solution was composed of 6.0 g/lsodium citrate dihydrate and 8.0 g/l sodium chloride inwater-for-injection (WFI).

The diluted blood solution was then concentrated back to its originalvolume by diafiltration through a 0.2 μm hollow fiber (Microgon KrosfloII microfiltration cartridge) diafilter. Concurrently, filtered isotonicsolution was added continuously, as makeup, at a rate equal to the rateof filtrate loss through the 0.2 μm diafilter. During diafiltration,components of the diluted blood solution which were significantlysmaller in diameter than RBCs, or are fluids such as plasma, passedthrough the walls of the 0.2 μm diafilter with the filtrate. RBCs,platelets and larger bodies of the diluted blood solution, such as whiteblood cells, were retained with continuously-added isotonic solution toform a dialyzed blood solution.

During RBC washing, the diluted blood solution was maintained at atemperature between approximately 10 to 25° C. with a fluid pressure atthe inlet of the diafilter between about 25 psi and about 30 psi toimprove process efficiency.

RBC washing was complete when the volume of filtrate drained from thediafilter equaled about 600% of the volume of blood solution prior todiluting with filtered isotonic solution.

The dialyzed blood solution was then continuously pumped at a rate ofapproximately 4 lpm to a Sharples Super Centrifuge, Model # AS-16,fitted with a #28 ringdam. The centrifuge was operating whileconcurrently being fed dialyzed blood solution, to separate the RBCsfrom the white blood cells and platelets. During operation, thecentrifuge rotated at a rate sufficient to separate the RBCs into aheavy RBC phase, while also separating a substantial portion of thewhite blood cells (WBCs) and platelets into a light WBC phase,specifically about 15,000 rpm. A fraction of the RBC phase and of theWBC phase were separately and continuously discharged from thecentrifuge during operation.

Following separation of the RBCs, the RBCs were lysed to form ahemoglobin-containing solution. A substantial portion of the RBCs weremechanically lysed while discharging the RBCs from the centrifuge. Thecell membranes of the RBCs ruptured upon impacting the wall of RBC phasedischarge line at an angle to the flow of RBC phase out of thecentrifuge, thereby releasing hemoglobin (Hb) from the RBCs into the RBCphase.

The lysed RBC phase then flowed through the RBC phase discharge lineinto a static mixer (Kenics 1/2 inch with 6 elements, Chemineer, Inc.).Concurrent with the transfer of the RBC phase to the static mixer, anequal amount of WFI was also injected into the static mixer, wherein theWFI mixed with the RBC phase. The flow rates of the RBC phase and theWFI into the static mixer are each at about 0.25 lpm.

Mixing the RBC phase with WFI in the static mixer produced a lysed RBCcolloid. The lysed RBC colloid was then transferred from the staticmixer into a Sharples Super Centrifuge (Model # AS-16, Sharples Divisionof Alfa-Laval Separation, Inc.) which was suitable to separate the Hbfrom non-hemoglobin RBC components. The centrifuge was rotated at a ratesufficient to separate the lysed RBC colloid into a light Hb phase and aheavy phase. The light phase was composed of Hb and also containednon-hemoglobin components with a density approximately equal to or lessthan the density of Hb.

The Hb phase was continuously discharged from the centrifuge, through a0.45 μm Millipore Pellicon Cassette, Cat # HVLP 000 C5 microfilter, andinto a holding tank in preparation for Hb purification. Cell stroma werethen returned with the retentate from the microfilter to the holdingtank. During microfiltration, the temperature within the holding tankwas maintained at 10° C. or less. To improve efficiency, when the fluidpressure at the microfilter inlet increased from an initial pressure ofabout 10 psi to about 25 psi, microfiltration was complete. The Hbmicrofiltrate was then transferred from the microfilter into themicrofiltrate tank.

Subsequently, the Hb microfiltrate was pumped through a 100,000Millipore Cat # CDUF 050 H1 ultrafilter. A substantial portion of the Hband water, contained in the Hb microfiltrate, permeated the 100,000Dalton ultrafilter to form a Hb ultrafiltrate, while larger cell debris,such as proteins with a molecular weight above about 100,000 Dalton,were retained and recirculated back into the microfiltrate tank.Concurrently, WFI was continuously added to the microfiltrate tank asmakeup for water lost in the ultrafiltrate. Generally, cell debrisinclude all whole and fragmented cellular components with the exceptionof Hb, smaller cell proteins, electrolytes, coenzymes and organicmetabolic intermediates. Ultrafiltration continued until theconcentration of Hb in the microfiltrate tank was less than 8grams/liter (g/l). While ultrafiltering the Hb, the internal temperatureof the microfiltrate tank was maintained at about 10° C.

The Hb ultrafiltrate was transferred into an ultrafiltrate tank, whereinthe Hb ultrafiltrate was then recirculated through a 30,000 DaltonMillipore Cat # CDUF 050 T1 ultrafilter to remove smaller cellcomponents, such as electrolytes, coenzymes, metabolic intermediates andproteins less than about 30,000 Daltons in molecular weight, and waterfrom the Hb ultrafiltrate, thereby forming a concentrated Hb solutioncontaining about 100 g Hb/l.

The concentrated Hb solution was then directed from the ultrafiltratetank onto the media contained in parallel chromatographic columns (2feet long with an 8 inch inner diameter) to separate the Hb by highperformance liquid chromatography. The chromatographic columns containedan anion exchange medium suitable to separate Hb from nonhemoglobinproteins. The anion exchange media was formed from silica gel. Thesilica gel was exposed to γ-glycidoxy propylsilane to form activeepoxide groups and then exposed to C₃ H₇ (CH₃)₂ NCl to form a quaternaryammonium anion exchange medium. This method of treating silica gel isdescribed in the Journal of Chromatography, 120:321-333 (1976).

Each column was pre-treated by flushing the chromatographic columns witha first buffer (Tris-acetate) which facilitated Hb binding. The pH ofthe buffer was about 9.0±0.1. Then 4.52 liters of the concentrated Hbsolution were injected into each chromatographic column. After injectingthe concentrated Hb solution, the chromatographic columns were thenwashed by directing buffer solutions through the chromatographic columnsto produce a stepped pH gradient of eluate from the columns. Thetemperature of each buffer during use was about 12.4° C. The bufferswere prefiltered through 10,000 Dalton ultrafiltration membrane beforeinjection onto the chromatographic columns.

In particular, a first buffer solution, 20 mM tris-hydroxymethylaminomethane acetate (Tris-acetate) (pH about 8.4 to about 9.4),transported the concentrated Hb solution in purified water (U.S.P.) intothe media in the chromatographic columns to bind the Hb. A secondbuffer, having a pH of about 8.3, then adjusted the pH withinchromatographic columns to elute contaminating non-hemoglobin componentsfrom the chromatographic columns, while retaining the Hb. Equilibrationwith the second buffer solution continued for about 30 minutes at a flowrate of approximately 3.56 lpm per column, or about 6.1 column volumes(11.7 void volumes). The eluate from the second buffer was discarded towaste. A third buffer solution, 50 mM Tris-acetate (pH about 6.5 toabout 7.5), then eluted the Hb from chromatographic columns as apurified hemoglobin product.

The Hb eluate was then directed through a sterile 0.22 μ Sartobran Cat #5232507 G1PH filter to a tank wherein the Hb eluate was collected. Thefirst 3-to-4% of the Hb eluate and the last 3-to-4% of the Hb eluatewere directed to waste.

The Hb eluate was further used if the elute contained less than 0.05EU/ml of endotoxin and contained less than 3.3 nmoles/ml phospholipids.To sixty liters of ultrapure elute, which had a concentration of 100 gHb/l, was added 9 l of 1.0 M NaCl, 20 mM Tris (pH 8.9) buffer, therebyforming an Hb solution with an ionic strength of 160 mM, to reduce theoxygen affinity of the Hb in the Hb solution. The Hb solution was thenconcentrated at 10° C., by recirculating through the ultrafilter,specifically a 10,000 Dalton Millipore Helicon, Cat # CDUF050G1 filter,until the Hb concentration was 110 g/l.

The Hb solution was then deoxygenated, until the pO₂ of the Hb solutionwas reduced to the level where HbO₂ content was about 10%, byrecirculating the Hb solution at 12 lpm, through a 0.05 μmHoechst-Celanese Corporation Cat # G-240/40) polypropylene microfilterphase transfer membrane, to form a deoxygenated Hb solution (hereinafter"deoxy-Hb"). Concurrently, a 60 lpm flow of nitrogen gas was directedthrough the counter side of the phase transfer membrane. Duringdeoxygenation, the temperature of the Hb solution was maintained betweenabout 19° C. and about 31° C.

Also during deoxygenation, and subsequently throughout the process, theHb was maintained in a low oxygen environment to minimize oxygenabsorption by the Hb and to maintain an oxygenated Hb (oxyhemoglobin orHbO₂) content of less than about 10% in the deoxy-Hb.

The deoxy-Hb 60 l, was then diafiltered through an ultrafilter with 180l of a storage buffer, containing 0.2 wt % N-acetyl cysteine, 33 mMsodium phosphate buffer (pH 7.8) having a pO₂ of less than 50 torr, toform a oxidation-stabilized deoxy-Hb. Prior to mixing with the deoxy-Hb,the storage buffer was depyrogenated with a 10,000 Dalton MilliporeHelicon, Cat # CDUF050G1 depyrogenating filter.

The storage buffer was continuously added at a rate approximatelyequivalent to the fluid loss across the ultrafilter. Diafiltrationcontinued until the volume of fluid lost through diafiltration acrossthe ultrafilter was about three times the initial volume of thedeoxy-Hb. The material may be stored at this point.

Prior to transferring the oxidation-stabilized deoxy-Hb into apolymerization apparatus, oxygen-depleted WFI was added to thepolymerization reactor to purge the polymerization apparatus of oxygento prevent oxygenation of oxidation-stabilized deoxy-Hb. The amount ofWFI added to the polymerization apparatus was that amount which wouldresult in a Hb solution with a concentration of about 40 g Hb/l, whenthe oxidation-stabilized deoxy-Hb was added to the polymerizationreactor. The WFI was then recirculated throughout the polymerizationapparatus, to deoxygenate the WFI by flow through a 0.05 μmpolypropylene microfilter phase transfer membrane (Hoechst-CelaneseCorporation Cat # 5PCM-108, 80 sq. ft.) against a counterflow ofpressurized nitrogen. The flow rates of WFI and nitrogen gas, throughthe phase transfer membrane, were about 18 to 20 lpm and 40 to 60 lpm,respectively.

After the pO₂ of the WFI in polymerization apparatus was reduced to lessthan about 2 torr pO₂, the polymerization reactor was blanketed withnitrogen by a flow of about 20 lpm of nitrogen into the head space ofthe polymerization reactor. The oxidation-stabilized deoxy-Hb was thentransferred into the polymerization reactor.

The polymerization was conducted in a 12 mM phosphate buffer with a pHof 7.8, having a chloride concentration less than or equal to about 35mmolar which was produced by mixing the Hb solution with WFI.

The oxidation-stabilized deoxy-Hb and N-acetyl cysteine weresubsequently slowly mixed with the cross-linking agent glutaraldehyde,specifically 29.4 grams of glutaraldehyde for each kilogram of Hb over afive hour period, while heating at 42° C. and recirculating the Hbsolution through a Kenics 11/2 inch static mixer with 6 elements(Chemineer, Inc.), to form a polymerized Hb (poly(Hb)) solution.

Recirculating the oxidation-stabilized deoxy-Hb and the glutaraldehydethrough the static mixer caused turbulent flow conditions with generallyuniform mixing of the glutaraldehyde with the oxidation-stabilizeddeoxy-Hb, thereby reducing the potential for forming pockets of deoxy-Hbcontaining high concentrations of glutaraldehyde. Generally uniformmixing of glutaraldehyde and deoxy-Hb reduced the formation of highmolecular weight poly(Hb) (having a molecular weight above 500,000Daltons) and also permitted faster mixing of glutaraldehyde and deoxy-Hbduring polymerization.

In addition, significant Hb intramolecular cross-linking resulted duringHb polymerization as an effect of the presence of N-acetyl cysteine uponthe polymerization of Hb.

After polymerization, the temperature of the poly(Hb) solution in thepolymerization reactor was reduced to a temperature between about 15° C.to about 25° C.

The poly(Hb) solution was then concentrated by recirculating thepoly(Hb) solution through the ultrafilter until the concentration of thepoly(Hb) was increased to about 85 g/l. A suitable ultrafilter is a30,000 Dalton filter (e.g., Millipore Helicon, Cat # CDUF050LT).

Subsequently, the poly(Hb) solution was then mixed with 66.75 g sodiumborohydride, to the poly(Hb) solution and then again recirculatedthrough the static mixer. Specifically, for every nine liters ofpoly(Hb) solution, one liter of 0.25 M sodium borohydride solution wasadded at a rate of 0.1 to 0.12 lpm.

Prior to adding the sodium borohydride to the poly(Hb) solution, the pHof the poly(Hb) solution was basified by adjusting pH to a pH of about10 to preserve the sodium borohydride and to prevent hydrogen gasformation. The pH of the poly(Hb) solution was adjusted by diafilteringthe poly(Hb) solution with approximately 215 l of depyrogenated,deoxygenated 12 mM sodium borate buffer, having a pH of about 10.4 toabout 10.6. The poly(Hb) solution was diafiltered by recirculating thepoly(Hb) solution from the polymerization reactor through the 30 kDultrafilter. The sodium borate buffer was added to the poly(Hb) solutionat a rate approximately equivalent to the rate of fluid loss across theultrafilter from diafiltration. Diafiltration continued until the volumeof fluid lost across the ultrafilter from diafiltration was about threetimes the initial volume of the poly(Hb) solution in the polymerizationreactor.

Following pH adjustment, sodium borohydride solution was added to thepolymerization reactor to reduce imine bonds in the poly(Hb) solution toketimine bonds and to form stable poly(Hb) in solution. During thesodium borohydride addition, the poly(Hb) solution in the polymerizationreactor was continuously recirculated through the static mixer and the0.05 μm polypropylene microfilter phase transfer membrane to removedissolved oxygen and hydrogen. Flow through a static mixer also providedturbulent sodium borohydride flow conditions that rapidly andeffectively mixed sodium borohydride with the poly(Hb) solution. Theflow rates of poly(Hb) solution and nitrogen gas through the 0.05 μmphase transfer membrane were between about 2.0 to 4.0 lpm and about 12to 18 lpm, respectively. After completion of the sodium borohydrideaddition, reduction continued in the polymerization reactor while anagitator contained therein rotated at approximately 75 rotations perminute.

Approximately one hour after the sodium borohydride addition, the stablepoly(Hb) solution was recirculated from the polymerization reactorthrough the 30,000 Dalton ultrafilter until the stable poly(Hb) solutionconcentration was 110 g/l. Following concentration, the pH andelectrolytes of the stable poly(Hb) solution were restored tophysiologic levels to form a stable polymerized Hb blood-substitute, bydiafiltering the stable poly(Hb) solution, through the 30,000 Daltonultrafilter, with a filtered, deoxygenated, low pH buffer containing 27mM sodium lactate, 12 mM NAC, 115 mM NaCl, 4 mM KCl, and 1.36 mM CaCl₂in WFI, (pH 5.0). Diafiltration continued until the volume of fluid lostthrough diafiltration across the ultrafilter was about 6 times thepre-diafiltration volume of the concentrated Hb product.

After the pH and electrolytes were restored to physiologic levels, thestable polymerized Hb blood-substitute was then diluted to aconcentration of 5.0 g/dl by adding the filtered, deoxygenated low pHbuffer to polymerization reactor. The diluted blood-substitute was thendiafiltered by recirculating from the polymerization reactor through thestatic mixer and a 100,000 Dalton purification filter against a filtereddeoxygenated buffer containing 27 mM sodium lactate, 12 mM NAC, 115 mMNaCl, 4 mM KCl, and 1.36 mM CaCl₂ in WFI, (pH 7.8). Diafiltrationcontinued until the blood-substitute contained less than or equal toabout 10% modified tetrameric and unmodified tetrameric species by GPCwhen run under dissociating conditions.

The purification filter was run under conditions of low transmembranepressure with a restricted permeate line. Following removal ofsubstantial amounts of modified tetrameric Hb and unmodified tetramericHb, recirculation of the blood-substitute continued through the 30,000Dalton ultrafilter until the concentration of the blood-substitute wasabout 130 g/l.

The stable blood-substitute was then stored in a suitable containerhaving a low oxygen environment and a low oxygen in-leakage.

EXAMPLE 2 Polymerized Hemoglobin Ananlysis

The endotoxin concentration in the hemoglobin product is determined bythe method "Kinetic/ Turbidimetric LAL 5000 Methodology" developed byAssociates of Cape Cod, Woods Hole, Mass., J. Levin et al., J. Lab.Clin. Med., 75:903-911 (1970). Various methods were used to test for anytraces of stroma for example, a precipitation assay, Immunoblotting, andenzyme-linked immunosorbent assay (ELISA) for a specific cell membraneprotein or glycolipid known by those skilled in the art.

Particulate counting was determined by the method "Particulate Matter inInjections: Large Volume Injections for Single Dose Infusions", U.SPharmacopeia, 22:1596, 1990.

To determine glutaraldehyde concentration, a 400 μl representativesample of the hemoglobin product was derivatized withdinitrophenylhydrazine and then a 100 μl aliquot of the derivativesolution was injected onto a YMC AQ-303 ODS column at 27° C., at a rateof 1 ml/min., along with a gradient. The gradient consisted of twomobile phases, 0.1% trifluoroacetic acid (TFA) in water and 0.08% TFA inacetonitrile. The gradient flow consisted of a constant 60% 0.08% TFA inacetonitrile for 6.0 minutes, a linear gradient to 85% 0.08% TFA inacetonitrile over 12 minutes, a linear gradient to 100% 0.08% TFA inacetonitrile over 4 minutes hold at 100% 0.08% TFA in acetonitrile for 2minutes and re-equilibrate at 45% of 0.1% TFA in water. Ultravioletdetection was measured at @360 nm.

To determine NAC concentration, an aliquot of hemoglobin product wasdiluted 1:100 with degassed sodium phosphate in water and 50 μl wasinjected onto a YMC AQ-303 ODS column with a gradient. The gradientbuffers consisted of a sodium phosphate in water solution and a mixtureof 80% acetonitrile in water with 0.05% TFA. The gradient flow consistedof 100% sodium phosphate in water for 15 minutes, then a linear gradientto 100% mixture of 80% acetonitrile and 0.05% TFA over 5 minutes, with ahold for 5 minutes. The system was then re-equilibrated at 100% sodiumphosphate for 20 minutes.

Phospholipid analysis was done by a method based on procedures containedin the following two papers: Kolarovic et al., "A Comparison ofExtraction Methods for the Isolation of Phospholipids from BiologicalSources", Anal. Biochem., 156:244-250, 1986 and Duck-Chong, C. G., "ARapid Sensitive Method for Determining Phospholipid Phosphorus InvolvingDigestion With Magnesium Nitrate", Lipids, 14:492-497, 1979.

Osmolarity was determined by analysis on an Advanced CryomaticOsmometer, Model #3C2, Advanced Instruments, Inc., Needham, Mass.

Total hemoglobin, methemoglobin and oxyhemoglobin concentrations weredetermined on a Co-Oximeter Model #482, from Instrumentation Laboratory,Lexington, Mass.

Na⁺, K⁺, Cl⁻, Ca⁺⁺, pO₂ concentration was determined by a NovastatProfile 4, Nova Biomedical Corporation, Waltham, Mass.

Oxygen binding constant, P₅₀ were determined by a Hemox-Analyzer, TCSCorporation, Southhampton, Pa.

Temperature and pH were determined by standard methods known by thoseskilled in the art.

Molecular weight (M.W.) was determined by conducting gel permeationchromatography (GPC) on the hemoglobin products under dissociatingconditions. A representative sample of the hemoglobin product wasanalyzed for molecular weight distribution. The hemoglobin product wasdiluted to 4 mg/ml within a mobile phase of 50 mM Bis-Tris (pH 6.5), 750mM MgCl₂, and 0.1 mM EDTA. This buffer serves to dissociate Hb tetramerinto dimers, that have not been cross-linked to other Hb dimers throughintramolecular or intermolecular crosslinks, from the poly(Hb). Thediluted sample was injected onto a TosoHaas G3000SW column. Flow ratewas 0.5 ml/min. and ultraviolet detection was recorded at 280 nm.

The results of the above tests on veterinary (OXYGLOBIN™) and human(HEMOPURE™b 2) Hb blood-substitutes, formed according to the method ofinvention, are summarized in Tables II and III, respectively.

                  TABLE II                                                        ______________________________________                                        PARAMETER           RESULTS                                                   ______________________________________                                        pH (18-22° C.)                                                                             physiologically accept                                       able pH                                                                      Endotoxin <0.5 EU/ml                                                          Sterility Test Meets Test                                                     Phospholipids.sup.a <3.3 nm/ml                                                Total Hemoglobin 12.0-14.0 g/dl                                               Methemoglobin <15%                                                            Oxyhemoglobin ≦10%                                                     Sodium, Na.sup.+ 145-160 mM                                                   Potassium, K.sup.+ 3.5-5.5 mM                                                 Chloride, Cl.sup.- 105-120 mM                                                 Calcium, Ca.sup.++ 0.5-1.5 mM                                                 Boron ≦10 ppm                                                          Osmolality 290-310 mOsm                                                       Glutaraldehyde <3.5 μg/ml                                                  N-acetyl-L-cysteine ≦0.2%                                              M.W. > 500,000 ≦15%                                                    Unmodified Tetramer ≦5%                                                Particulate Content ≧ 10μ <12/ml                                    Particulate Content ≧ 25μ <2/ml                                   ______________________________________                                         .sup.a measured in Hb before polymerization                              

                  TABLE III                                                       ______________________________________                                        PARAMETER             RESULTS                                                 ______________________________________                                        pH (18-22° C.) Physiologically                                            acceptable pH                                                                Endotoxin <0.5 EU/ml                                                          Sterility Test Meets Test                                                     Phospholipids.sup.a <3.3 nm/ml                                                Total Hemoglobin 12.0-14.0 g/dl                                               Methemoglobin <15%                                                            Oxyhemoglobin ≦10%                                                     Sodium, Na.sup.+ 145-160 mM                                                   Potassium, K.sup.+ 3.5-5.5 mM                                                 Chloride, Cl.sup.- 105-120 mM                                                 Calcium, Ca.sup.++ 0.5-1.5 mM                                                 Boron ≦10 ppm                                                          Osmolality 290-310 mOsm                                                       Glutaraldehyde <3.5 μg/ml                                                  N-acetyl-L-cysteine ≦0.2%                                              M.W. > 500,000 ≦15%                                                    M.W. ≦ 65,000 ≦10%                                              M.W. ≦ 32,000 ≦5%                                               Particulate Content ≧ 10μ <12/ml                                    Particulate Content ≧ 25μ <2/ml                                   ______________________________________                                         .sup.a measured in Hb before polymerization                              

EXAMPLE 3 Determination of In Vitro Oncotic Effects in Canines

The purpose of this study was to determine the in vivo oncotic effects,specifically the volume of water drawn into the intravascular space pergram of hemoglobin administered, of veterinary (OXYGLOBIN™) Hbblood-substitute in splenectomized beagle dogs by measuring theexpansion of plasma volume following a toploading dose. In addition, acomparable dose of (RHEOMACRODEX™-Saline), manufactured by Pharmacia,which is 10% Dextran 40 and 0.9% saline, was also determined.

Two dogs were entered into this study after a routine health screeningand an acclimatization period of at least four weeks. The dogs weresplenectomized at least 3 days before treatment. They werepre-anesthetized, with a combination of atropine and meperidine HCl, andanesthetized via inhalation of isofluorane. Lactated Ringer's solutionwas infused at 10-20 ml/kg/hr during the surgical procedure.

The dogs received the Hb blood-substitute (40 ml/kg) at 20 ml/kg/hr viaa disposable cephalic catheter. Hematocrit was measured pre-dosing andat 1/4, 1/2, 1, 2, 3, 4 hours post-dosing or longer until the nadir ofthe hematocrit was established.

The dogs were splenectomized to ensure a constant plasma volume and RBCmass to allow accurate measurement of the change in plasma volumefollowing dosing.

Calculation of the change in plasma volume was made using the followingequation: ##EQU1## where PV is the plasma volume, Hct₁ is the initialhematocrit, and Hct₂ is the final hematocrit. This calculation was basedon the change in hematocrit, assuming that the number of RBC's withinthe circulating blood volume and mean corpuscular volume remainedconstant.

As shown in Table IV, the nadir of the hematocrit occurred two hourspost-dosing in both dogs. The mean corpuscular volume (MCV) remainedstable throughout the study.

                  TABLE IV                                                        ______________________________________                                               Hematocrit (%)                                                                              MCV (fL)                                                 Time (Hour)                                                                            Dog 3503C Dog 14 Male                                                                             Dog 3503C                                                                             Dog 14 Male                              ______________________________________                                        0        46        55        67.6    67.2                                       1/4  41 50 68.1 67.7                                                          1/2  37 48 67.5 67.2                                                          1 35 41 68.6 67.9                                                             2 31 37 68.1 67.1                                                             3 33 39 66.8 66.1                                                             4 32 40 66.3 65.4                                                           ______________________________________                                    

The volume of fluid drawn intravascularly post dosing was 6 ml/ghemoglobin and 9 ml/g hemoglobin for dogs 3503C and 14 male,respectively. The dose of synthetic colloid solution(Rheomacrodex®-Saline) was calculated based on a dose that causes asimilar oncotic effect. Rheomacrodex draws approximately 22 ml fluidfrom the interstitium per gram administered intravenously.

The calculated comparable dose of Rheomacrodex was 14 ml/kg and 7 ml/kgfor 30 ml/kg and 15 ml/kg Hb blood-substitute respectively.

The volume of fluid drawn intravascularly by (Oxyglobin™) Hbblood-substitute was 8 ml H₂ O/gram hemoglobin. Since the volume of thedose was 30 ml/kg, and the concentration of hemoglobin in the dose was13 g/dl, the total amount of hemoglobin per dose was 3.9 g/kg and thetotal volume of fluid drawn into the intravascular space/dose by the Hbblood-substitute was 31.2 ml

The synthetic colloid solution draws in about 22 ml of water/gram ofDextran. The total amount of Dextran in the colloid solution percomparable dose of Hb blood-substitute is 1.4 g. Thus, the total volumeof fluid drawn into intravascular space/comparable dose of colloidsolution is 14 ml.

EXAMPLE 4 Canine Dose Response Study

This study was conducted to determine the drug effect and dose responseof veterinary (OXYGLOBIN™) Hb blood-substitute of this invention, ascompared to a synthetic colloid solution, of (RHEOMACRODEX™-Saline,Pharmacia) which is 10% Dextran 40 and 0.9% saline, with respect toarterial oxygen content relative to canine red blood cell hemoglobin andoxygen delivery in splenectomized beagle dogs 60 minutes and 24 hoursfollowing acute normovolemic hemodilution.

Acute normovolemic hemodilution is an experimental model that mimics aclinical condition of anemia due to surgical blood loss. Severe anemia(Hct=9%, Hb=3 g/dl) was produced by this method to cause an absoluterequirement of oxygen carrying support. Oxygen delivery and oxygencontent decreased precipitously with the massive bleeding.

In developing the normovolemic hemodilution model, it was found thattreatment to restore oxygen delivery either by volume expansion alone,as was done for the control dogs, or by volume expansion in conjunctionwith an increase in the arterial oxygen content, as occurred for thedogs treated with hemoglobin solution, had to occur within approximately10 minutes of reaching a hematocrit of 9% to avoid irreversibledecreases in blood pressure and cardiac output which then resulted indeath.

Two of 12 control dogs in this study died during or following dosingeven though their vascular volume was expanded with Dextran 40 solutionwithin 5 minutes of reaching the targeted hematocrit. The death of thesedogs is a reflection of the severity of the experimental model which inturn portrays the clinical condition of severe acute blood loss.

Thirty dogs were entered into this study after a routine healthscreening and acclimatization period of at least four weeks. Treatmentwas staggered using three replicates of dogs (A, B and C), eachreplicate containing one dog/sex/group. Dogs were randomly assigned tothe 5 groups (6 dogs/group of 3 males and 3 females) 32 days before thefirst day of treatment. Dogs were assigned to their respective groups byblock randomization based on body weight using a method which ensuredequal distribution among groups. Males and females were randomizedseparately. Any dog with unacceptable pretreatment data, such asabnormal clinical signs or clinical pathology data, was replaced by aspare dog maintained under the same environmental conditions.

The test/control articles were administered by a single intravenousinfusion. The rate of infusion was controlled by an infusion pump. Theactual volume infused per hour depended upon the most recent body weightof each of the dogs.

The highest dose of hemoglobin solution was based upon the safe upperlimit of acute cardiovascular effects due to volume expansion innormovolemic dogs. The mid-range dose was chosen to define the shape ofthe dose response curve. The lowest dose was based on the lower limit ofclinically relevant dosing as defined by volume and hemodynamic effectsin the dog.

Each dog was splenectomized at least 7 days before treatment to avoideffects on the experimental model of an increased circulatory RBC massdue to splenic contraction. On the day of treatment with hemoglobinsolution, each dog was anesthetized by inhalation of isoflurane andmechanically ventilated using room air with a tidal volume of 20-25ml/Kg. The rate of ventilation was adjusted during the procedure tomaintain arterial pCO₂ at approximately 40 mmHg. The end-expiredconcentration of isoflurane was measured and controlled to provide avalid comparison of anesthetic plane from dog to dog. The dogs wereinstrumented for monitoring of hemodynamic function and oxygen transportparameters. Placement of a flow-directed catheter in the pulmonaryartery was confirmed by analysis of pressures and pressure tracings. Adual-lumen catheter, with thermodilution cardiac output capability, wasplaced in the femoral artery to provide an arterial line for bloodpressure monitoring and blood withdrawal. A catheter was placed in thecephalic vein, or other vein if required, for volume replacement andtest/control article administration.

Each dog received an intramuscular injection of antibiotics once daily(Procaine penicillin G) prophylactically for one day prior to surgery,on the day of surgery and for 3 days following the splenectomy.V-Sporin, a topical antibiotic (Polymyxin B, Bacitracin, Neomycin) wasapplied to the surgical site once daily, as needed.

Following instrumentation, hemodynamic stabilization to reach a pCO₂ ofapproximately 40 mm Hg and collection of baseline measurements wereperformed. A model of acute normovolemic hemodilution was then producedby bleeding the dogs and simultaneously replacing approximately 1.6 to2.3 times the volumes withdrawn with Lactated Ringer's Solution tomaintain isovolemic status. Isovolemic status was achieved bymaintaining pulmonary artery wedge pressure at approximately baselinevalues. The blood withdrawal/volume replacement took approximately 45 to90 minutes until the hemoglobin concentration was approximately 30 g/l(3.0 g/dl). Lactated Ringer's Solution was infused rapidly using agravity intravenous set and a pressure cuff around the infusion bag. Ifthe arterial systolic blood pressure was ≦50 mmHg for more than 5minutes following the induction of acute anemia and prior to the startof dosing, the dog was rejected and replaced by a spare dog maintainedunder the same environmental conditions.

Doses of colloid control and hemoglobin solution were administered asstated in Table V. Hemodynamic measurements were performed pre-bleed,pre-dose, immediately following dosing, and at 60 minutes and 24 hoursfollowing dosing. After the 60 minute measurement, the dog recoveredfrom anesthesia and was instrumented again for hemodynamic measurements,performed at 24 hours following dosing.

                  TABLE V                                                         ______________________________________                                                         Dose Volume                                                                              Dose Rate                                                                            Animals/Group                              Group Test Article                                                                             ml/Kg      ml/Kg/h                                                                              Males Females                              ______________________________________                                        1     Colloid control                                                                          14         20     3     3                                       (mid dose)                                                                   2 Colloid control  7 20 3 3                                                    (low dose)                                                                   3 Hb blood- 15 20 3 3                                                          substitute                                                                    (low dose)                                                                   4 Hb blood- 30 20 3 3                                                          substitute                                                                    (mid dose)                                                                   5 Hb blood- 45 20 3 3                                                          substitute                                                                    (high dose)                                                                ______________________________________                                    

All hemodynamic parameters were statistically analyzed by eitheranalysis of variance (ANOVA) or analysis of covariance (ANCOVA) witheither the pre-bleeding or pre-dosing value as the covariate. Specificlinear contrasts were constructed to test for the effects of volume ofthe solution administered, the effect of Hb blood-substitute (drugeffect), and the dose response of the Hb blood-substitute (dose effect).These tests were performed only for parameters for which the differenceamong experimental groups was statistically significant at the 0.05level. Comparisons of specified variables at selected time points wereperformed by paired t-tests in each group.

Arterial oxygen content was one criterion of efficacy in this study.Arterial oxygen content is a measure of the oxygen carrying capacity ofcellular and plasma hemoglobin and dissolved oxygen in the plasma. Inthe absence of plasma hemoglobin, arterial oxygen content is calculatedfrom the amount of oxygen carried by saturated cellular hemoglobin andthe partial pressure of inspired oxygen. Because plasma hemoglobin wasexpected to contribute significantly to oxygen content in this study,oxygen content was measured directly using a LexO2Con-K instrument(Chestnut Hill, Mass.). Oxygen enriched air was not administered duringthe experiment because it was unnecessary and to avoid the confoundingeffects of an increased inspired oxygen concentration on the measurementof arterial oxygen content.

Mean arterial and venous oxygen contents decreased approximately fourand eight times, respectively in all groups following induction ofanemia. Arterial oxygen content increased significantly 60 minutesfollowing dosing compared to pre-dosing values in all Hbblood-substitute treated groups and remained significantly increased at24 hours following dosing in the mid and high dose groups. Arterial orvenous oxygen content did not change following dosing in either controlgroup.

As shown in FIG. 2, arterial oxygen content was significantly increasedin Hb blood-substitute treated groups compared to control groups at 60minutes and 24 hours following dosing. A linear dose response was seenat 60 minutes and 24 hours following dosing. A significant volume effectwas detected for arterial oxygen content 60 minutes following dosing.

Venous oxygen content also significantly increased in Hbblood-substitute treated groups compared to controls at 60 minutes and24 hours following dosing. The increase showed a linear dose response at60 minutes following dosing but not at 24 hours.

The dose effect observed for Hb blood-substitute treated groups inarterial-venous (A-V) oxygen content difference at 60 minutes followingdosing was attributed to significant volume effects based on the absenceof a drug effect and similar observations of volume effects in controlgroups at 60 minutes following dosing. Hb blood-substitute treatedgroups showed a significant increase in A-V oxygen difference at 24hours compared to colloid controls, with a significant linear doseresponse. The A-V difference must be interpreted in view of the cardiacoutput. At 24 hours following dosing, the A-V difference in the controlgroups was significantly lower than that of the Hb blood-substitutetreated groups. One possible explanation for this difference is that thecontrol group dogs had to rely on a higher cardiac output to meet theoxygen consumption needs of peripheral tissues. The Hb blood-substitutetreated groups maintained a large enough A-V difference at 24 hoursfollowing dosing to meet peripheral tissue needs without cause for anincreased cardiac output.

In addition to arterial oxygen content, total arterial oxygen contentnormalized relative to the contribution of canine RBC hemoglobin (CaO₂/g RBC Hb) was examined in this study. This comparison was made todemonstrate differences in arterial oxygen content among dosing groupssince the RBC hemoglobin was constant in all groups. The potentialcorrelation of plasma or total hemoglobin concentration and arterialoxygen content would provide a useful clinical measure of efficacy. Asshown in FIG. 3, at 60 minutes and 24 hours following dosing, all Hbblood-substitute treated groups (except the low dose group at 24 hours)showed a significant increase in CaO₂ /g RBC hemoglobin to pre-dosingvalues. Arterial oxygen content relative to that contributed by RBChemoglobin did not differ significantly in the colloid controls betweenpre-dose and 60 minutes or 24 hours following dosing.

Total arterial oxygen content relative to that contributed by red bloodcell hemoglobin significantly increased in Hb blood-substitute treatedgroups compared to colloid controls at 60 minutes following dosing witha significant linear dose response. A significant dose effect alsooccurred at 24 hours following dosing with a significant linear doseresponse, but the drug effect was not quite significant (P<0.06).

Oxygen delivery was another criterion of efficacy. Oxygen delivery iscalculated based on arterial oxygen content and cardiac output.Therefore, oxygen delivery is affected by all the physiologic factorswhich influence cardiac output. The control chosen for this study wasthe synthetic colloid (RHEOMACRODEX™-Saline, Pharmacia) which is 10%Dextran 40 and 0.9% saline, as it expands intravascular volume and isnot known to carry oxygen. The control provided a comparison ofequivalent volume expansion to the colloidal properties of thehemoglobin in Hb blood-substitute.

Because each dose of Hb blood-substitute was expected to demonstrate adistinct volume effect, two doses of dextran solution were used ascontrols for the volume effect so the data would reflect only the drugeffect of different doses. This comparison was made for the low and middoses. The doses of colloid controls were selected based on those dosesof Dextran 40 which provided an equivalent comparison of the in vivooncotic effects of the low and mid-dose test articles, as determinedfrom the results of Example 2.

The volume effect was defined statistically using the difference inmeans between the colloid mid dose (14 ml/kg) and the colloid low dose(7 ml/kg). The drug effect was determined by comparing each Hbblood-substitute treated group to its corresponding colloid control. Alinear dose response was established when a statistically significantdifference was seen between the low and high dose Hb blood-substitutetreated groups.

Oxygen delivery was calculated according to the equation: D0₂ =CO×CaO₂×10/kg which CO is the cardiac output and CaO₂ is the arterial oxygencontent. As expected, following induction of anemia in all treatmentgroups, a two to three fold mean decrease in DO₂ occurred in all groups.The oxygen content decreased sufficiently that the maintenance ofbaseline oxygen consumption had to result from an increase in cardiacoutput and increased extraction of oxygen, resulting in a lower venousoxygen content. As shown in FIG. 4, oxygen delivery increasedapproximately 30% in the low dose Hb blood-substitute treated group andgreater than 100% in the mid and high Hb blood-substitute treated groupsat 60 minutes following dosing compared to pre-dosing values. Thedifference was significant for all three dosing groups (p<0.05). Thecontrol groups showed no significant differences over this time. At 60minutes following dosing, DO₂ differed significantly among all groupswith significant drug and dose effects with a linear dose response. At24 hours, no difference in oxygen delivery was noted among groups. Theimprovement in oxygen delivery at 60 minutes following dosing for all Hbblood-substitute treated groups, as compared to their correspondingcolloid controls, was due primarily to a dose related increase inarterial oxygen content in addition to a modest increase in cardiacoutput.

Oxygen consumption was calculated according to the equation: VO₂=CO×CaO₂ ×10 kg. A two to three fold mean decrease in DO₂ occurred inall groups following the induction of anemia. No statisticallysignificant differences were noted among Hb blood-substitute treated orcontrol groups or within a group when comparing pre-dosing topost-dosing values.

The Oxygen Extraction Ratio (VO₂ /DO₂) for all groups showed anapproximately three fold increase following induction of anemia. Oxygenextraction ratios were significantly decreased in a dose dependentmanner in all Hb blood-substitute treated groups at 60 minutes followingdosing compared to control groups. No significant differences were notedbetween Hb blood-substitute treated and control groups at 24 hoursfollowing dosing.

Mean cardiac output increased between 10% and 39% in all groupsfollowing induction of anemia. Cardiac output was significantlyincreased at 24 hours following dosing compared to pre bleeding valuesin the colloid control groups but not in the Hb blood-substitute treatedgroups. A significant volume effect which contributed to significantdifferences in cardiac output between colloid low and mid dose groupswas evident at 60 minutes post-dosing. The increase in cardiac outputwas likely related to an increased stroke volume due to expansion of theintravascular volume following dosing or increased sympathetic tone dueto the stress of severe anemia. A significant dose response between Hbblood-substitute low and high dose groups was apparent at 60 minutes,but not at 24 hours following dosing. No difference in cardiac outputbetween Hb blood-substitute treated and colloid control groups was seenat 60 minutes or 24 hours following dosing.

Pulmonary artery wedge pressure (PAWP) did not change significantlyduring the induction of anemia. PAWP decreased significantly in the lowdose colloid group and remained unchanged in the mid dose colloid group60 minutes following dosing compared to pre dosing values. The PAWP inthe mid and high dose Hb blood-substitute treated groups increasedsignificantly in a linear dose response compared to pre-dosing values at60 minutes following dosing. The increased PAWP reflected a dosedependent increase in intravascular volume at 60 minutes followingdosing. No significant drug effect was detected between Hbblood-substitute treated and control groups at 60 minutes or 24 hoursfollowing dosing. A significant volume effect was detected in thecolloid control groups at 60 minutes following dosing.

Systolic, diastolic and mean arterial blood pressure decreasedsignificantly in all groups following induction of anemia, thenincreased significantly immediately following dosing. The decrease insystolic arterial blood pressure after the induction of anemia waslikely related to a decrease in peripheral vascular resistance due todecreased blood viscosity, a consequence of anemia. At 60 minutesfollowing dosing, the systolic, diastolic, and mean arterial bloodpressures of both colloid control groups did not differ significantlyfrom pre-dosing values. The systolic, diastolic, and mean pressures ofthe low dose colloid control increased significantly compared topre-dosing values at 24 hours following dosing. In contrast, theincrease in systolic, diastolic, and mean pressures was statisticallysignificant in all Hb blood-substitute treated groups at 60 minutes and24 hours following dosing compared to pre-dosing values. The systolic,diastolic and mean blood pressures of Hb blood-substitute treated groupswere significantly higher than corresponding colloid control groups at60 minutes following dosing, but not at 24 hours.

Significant increases in systolic, diastolic and mean pulmonary arterialpressures were observed in the mid and high dose Hb blood-substitutetreated groups 60 minutes post dosing compared to pre dosing values. Theincreases persisted at 24 hours post-dosing in the mid Hbblood-substitute treated group for pulmonary diastolic arterialpressure. Additionally the low-dose colloid group showed a statisticallysignificant increase at 24 hours post-dosing compared to pre-dosingvalues for mean pulmonary artery pressure. This increase was consideredclinically significant. The increases in systemic arterial systolic anddiastolic blood pressure 60 minutes following dosing of Hbblood-substitute, compared to pre-dosing values, were a direct drugeffect of the Hb blood-substitute. The diastolic pressure remainedunchanged in the colloid control groups which was probably a result of adecreased peripheral vascular resistance.

No significant differences were found between Hb blood-substitutetreated and control groups for pulmonary systolic arterial pressure ateither 60 minutes or 24 hours post-dose. In contrast, pulmonarydiastolic and mean arterial pressures were significantly different withregard to volume, drug, and dose effects at 60 minutes post dosing, butnot at 24 hours.

Total hemoglobin decreased approximately four times or greater withbleeding. Hb blood-substitute treated groups showed a dose dependentincrease in total hemoglobin compared to corresponding colloid controlgroups at 60 minutes and 24 hours following dosing.

Plasma hemoglobin concentrations significantly increased in a dosedependent manner in Hb blood-substitute treated groups compared tocorresponding colloid control groups at 60 minutes and 24 hoursfollowing dosing. The increases in plasma and total hemoglobinconcentrations following dosing in all Hb blood-substitute treatedgroups, as compared to their corresponding colloid controls, wereattributable to the hemoglobin content of Hb blood-substitute. The dosedependent significant increase persisted for 24 hours, correlating withthe persistent increase in arterial oxygen content.

In summary, the response to treatment with the Hb blood-substitute waslinear, i.e., at 60 minutes following dosing, the higher the dose of Hbblood-substitute the greater the improvement in oxygen delivery andhemodynamics compared to corresponding colloid controls. Sustainedarterial oxygen content and normal clinical signs, while breathing roomair, support a beneficial biological effect of Hb blood-substitutelasting 24 hours in the 30 ml/kg and 45 ml/kg dose Hb blood-substitutetreated groups. The clearance of Hb blood-substitute likely accounts forthe changes seen in oxygen delivery and hemodynamic effects at 24 hoursfollowing dosing. In conclusion, results from this study supportselection of a dose ranging from 30 to 45 ml/kg. Both of these dosinggroups showed statistically significant differences from correspondingcolloid control groups in the parameters of efficacy and the doseresponse was linear.

The clinical rationale of this dosing range is based on the fact that aseverely anemic dog (e.g., hematocrit <15% with marked clinical signs)would benefit from a higher dose as demonstrated by the linear doseresponse of improved arterial oxygen content and oxygen delivery.However, a more conservative dose would be indicated for a dog which maybe predisposed to intravascular volume overload. The dose dependenttransient increase in pulmonary artery wedge pressure and pulmonaryarterial pressures seen 60 minutes following dosing in Hbblood-substitute treated groups would limit the use of a higher dose inthis population of dogs. Therefore a dosing range of 30-45 ml/kg wouldbe effective in a broad population of dogs in which the degree of anemiaand intravascular volume status are defined.

EXAMPLE 5 Human Dose Response Study

This study was conducted to evaluate the safety and tolerance ofincreasing rates of intravenous administration of Hb blood-substitute(hereinafter HBOL) upon hemodynamic, neuroendocrine and hematologicparameters in humans. The test subjects were normal healthy adult males(70-90 kg) between the ages of 18-45 years. During the study, the testsubjects were on controlled isocaloric diets of 55% carbohydrates, 30%fat (polyunsaturated to saturated fat ratio of 2:1), 15% protein and 150mEq of sodium per day. Fluid intake was at least 3000 mls/day withcaffeine containing beverages avoided. Also concomitant use ofmedication was avoided. Further no alcohol or tobacco were used by thetest subjects during the study.

The 12 subjects studied, were divided into three test groups. In eachtest group, three subjects received HBOL and one served as a control,receiving Ringer's lactate. Each test group had different rates of HBOLinfusion. The study was conducted as a single-blind, rate escalationstudy over a thirty day interval.

On Day 1 of the study, during the inpatient phase, each subject had asmall gauge arterial catheter inserted in the radial artery of thenon-dominant hand. The insertion location was cleansed with anantiseptic solution (alcohol and/or iodine) and then a small amount of1%-2% Lidocaine anesthetic solution was subcutaneously injected over thesite of the radial artery. The arterial catheter was inserted to monitorblood pressure and to facilitate blood gases evaluations. One to twohours later, all subjects had one large-bore intravenous catheter(16-gauge needle in antecubital fossa) placed in a vein in one arm. Eachsubject then had a phlebotomy of 750 ml (1.5 units) of whole blood drawnin less than 15 minutes, which was then followed with isovolemichemodilution by the infusion of 2250 ml of Ringer's lactate over a 2hour period.

Forty-five grams (346 ml) of HBOL were then intravenously infused usingsterile technique, in series through a standard 80 micrometer bloodfilter, a 5 micrometer filter, and the large-bore intravenous catheterin the arm vein, into each subject in the test groups 1, 2 and 3 at therates of 0.5 gm/minute, 0.75 gm/minute and 1.0 gm/minute, respectively.

Simultaneously, each subject had invasive monitoring by radial arterycatheter, serial pulmonary function tests, cardiac function evaluationand multiple hematology, chemistry and urinalysis laboratory tests whichwere routinely and frequently performed over the first 28 hours aftercommencing HBOL infusion.

Subsequently, in the outpatient phase (Days 2-29), laboratory studies,vital signs, ECGs and medical events were taken daily for the first fourdays post-discharge and then on a weekly basis for a month.

Hemodynamics were remarkable for generally higher values for systolic,diastolic and mean arterial pressure in the HBOL-treated groups (afterinfusion) than controls during Day 1. Although there was markedvariability in the blood pressure data commensurate with patientactivity (e.g., during meals or when using the bathroom) and diurnalrhythm, HBOL-treated subjects generally had values for the systolicblood pressure (about 5-15 mm Hg), diastolic blood pressure (about 5-10mm Hg) and mean arterial pressure (about 10 mm Hg) greater than controlsonly during the course of Day 1. Values tended to reach peak effectsbetween Hours 8-12 with return to baseline during sleep and upon removalof the arterial catheter. Pulse was generally about 10 beats lower inall HBOL-treated groups compared to controls during Day 1. The nadir ofpulse decline was seen within the first 15 minutes of the infusion.Values were similar in all test groups after hour 24.

Cardiac index declined about 1-2 1/min/m² during the first hour ofinfusion remained up to 1 1/min/m² lower than controls through hour 4,and then it returned to baseline by hour 4. Cardiac index also increasedduring times of patient activity (as above).

Total peripheral resistance paralleled blood pressure changes, however,values returned to baseline within two hours. The transient increase insystemic blood pressure with an increase in total peripheral resistanceand decrease in cardiac index is not unexpected. It is important to notethat there was no difference in the rate of administration and themagnitude of these hemodynamic responses and that no intervention wasindicated.

The pulmonary function tests (including multiple determinations ofspirometry and lung volumes) and arterial blood gas measurements wereunremarkable. What was noteworthy was the enhanced diffusion capacitythat was seen in the HBOL-treated groups. The 10-15% increase indiffusion capacity was statistically significant compared to a 10%decrease in the controls for up to 24 hours. These findings areparticularly important because of the magnitude of phlebotomy andhemodilution that all of the groups underwent.

In hematological studies, other than the expected, transient decline inhemoglobin, hematocrit, red cell count and serum proteins with thephlebotomy and hemodilution procedures, the hematology and serumchemistry laboratory tests were unremarkable. Exceptions were serum ironand ferritin which showed peak values by Hours 6 and 48, respectively,after HBOL was given.

The serum chemistry measurements were unremarkable, with the exceptionof one subject (#10) who had transient increases in serum transaminasesand lipase. It is important to note that this subject did not have anyclinically significant concomitant medical events (e.g., dysphagia orabdominal pain) commensurate with the time of the elevation of theseenzymes. The exact etiology of these laboratory abnormalities isunclear, but previous studies suggest that transient subclinical spasmof the sphincter of Oddi or other portions of the hepatobiliary andpancreatic ductal systems may be involved. It is important to note thatthese changes were transient (and unaccompanied by abdominal discomfort)and without apparent sequelae. No significant change was noted inSubject #10's post-dose ultrasound of the gall bladder.

Urinalysis was unremarkable throughout the study. There was nodetectable urinary hemoglobin in the subjects during the study. Inaddition, creatinine clearance was slightly higher, as expected, duringthe hemodilution period), urinary adenosine deaminase binding protein,electrolytes (sodium, potassium, chloride), iron, microalbumin, NAG(N-acetyl-beta-glucosaminidase) and urinary urea nitrogen wereunremarkable.

No apparent changes in the majority of the pharmacokinetic parameterswere observed as a function of administration rate. Sequential bloodspecimens and cumulative urine specimens were collected prior to andfollowing initiation of infusion of HBOL for size exclusion (gelfiltration) chromatographic (SEC) analysis of total hemoglobin andapparent molecular weight fractions of hemoglobin. Only sporadic plasmadimmer fraction concentrations were observed precluding anypharmacokinetic analysis. The only statistically significant differences(p<0.05) were observed in the tetramer volume of distribution (decreaseswith increases in rate), tetramer maximum concentration achieved(increases with increases in rate) and the time of the tetramer maximumconcentration occurrence (decreases with increases in rate).

The observed medical events were consistent with expected findingsrelated to phlebotomy (e.g., vasovagal episode), multiple pulmonaryfunction tests (aerophagia, eructation or abdominal "gas"), arterialline insertion (e.g., pain or tingling over the site), or abdominaldiscomfort (e.g., associated with the ingestion of the iron supplement).Although there seemed to be a background of nonspecific, transientabdominal "gas." there were no cases of overt abdominal pain ordysphagia. In addition there was no correlation of these symptoms withany alterations in serum transaminases or lipase.

In summary, HBOL was well tolerated. Although there were small transientincreases in blood pressure and total peripheral resistance withcommensurate decline in cardiac index during the first two hours of theinfusion, the hemodynamics were unremarkable. The increase in diffusioncapacity was significantly higher in the HBOL-treated groups thancontrols during the first 24 hours.

EXAMPLE 6 Effects of HBOL on Humans in Graded Bicycle Exercise Testing

This study was conducted to evaluate the exercise capacity of subjectsgiven autologous transfusion of HBOL. Specific endpoints includedpulmonary function (e.g., diffusion capacity and lactic acid levels andpO₂), hemodynamics (e.g,, heart rate, cardiac index and blood pressure)and exercise tolerance (e.g., duration, workload and anaerobicthreshold). The subjects were six normal healthy male humans, ages 18-45years. One subject was replaced in the study due to failure to obtainthe volume of phlebotomy in less than 15 minutes. The study wasconducted as a randomized, single-blind, two-way crossover study.

All subjects had phlebotomy of 750 ml followed by Ringer's Lactate [3:1]and either an autologous transfusion (ATX) or 45 gms of HBOL. The ATX orHBOL was given at 0.5 gm/min for 90 minutes. Bicycle exercise stresstests were done on the day prior to phlebotomy and approximately 45minutes after the infusion of ATX or HBOL. The same procedures wererepeated one week later and subjects were crossed over to the oppositetreatment.

On the day of dosing (Days 1 and 8), all subjects had insertion of anarterial line in one radial artery, attachment to cardia telemetry andimpedance cardiography and then phlebotomy (PBX) of 750 ml of wholeblood (<15 minutes). This was followed by an infusion of 2250 ml ofRinger's lactate (RL) over two hours (the isovolemic hemodilutionphase). Subjects then received either HBOL (45 gms [about 346-360 ml] ata rate of 0.5 gm/min over 90 minutes) or an ATX (110-120 gms ofhemoglobin [about 750 ml] at the same rate and duration a the HBOL). TheBEST was done about 45 minutes after the end of either infusion. Serialmeasurements of arterial blood gases, hematology, chemistry and urinetests were made intensively during the 24 hour period on Days 1 and 8.Serial follow-up was done on an outpatient basis between the dosing andfor one month after all dosing was complete.

Subjects were able to exercise to similar levels during HBOL and ATXperiods. The oxygen uptake (VO₂) and carbon dioxide production (VCO₂) atanaerobic threshold were nearly identical. The actual workload in METS,watts, pulse (as a % of maximum pulse), time to anaerobic threshold,tidal volume (VT) and minute ventilation (VE) were also similar.Arterial blood gas values were similar during the HBOL and ATX periods.The small reductions in pH and bicarbonate with increase in lactic acidis consistent with expected findings at anaerobic threshold. The resultsof these bicycle tests showed that exercise capacity (defined as timeand workload to reach anaerobic threshold) was similar at baseline andafter infusions of either autologous transfusion or HBOL. Specifically,hemodynamics were remarkable for slightly higher values (˜5 mmHg) duringthe HBOL period for systolic, diastolic and mean arterial pressure.Commensurate with the increase in blood pressure was an increase intotal peripheral resistance, generally within the first 4 hours. Cardiacindex declined during the HBOL period (˜0.5 1/min/M²). Pulse was about5-10 beats lower during the HBOL than the ATX period. These findingshave been observed in the HBOL studies and have been of little clinicalconcern.

Pulmonary function tests were unremarkable except for a 14% increaseabove baseline in diffusion capacity after the ATX and HBOL infusions.Subjects were able to achieve similar exercise capacity during HBOL andATX periods. Arterial blood gas measurements during peak exercise(anaerobic threshold) were similar in both periods, but arterial pO₂tended to be higher during the HBOL period. Plasma lactic acid levelswere lower during the HBOL than ATX period. Resting metabolic artmeasurements indicated that oxygen consumption, carbon dioxideproduction and metabolic energy expenditure were greater during the HBOLthan ATX period. The comparison as mentioned above is roughly one gramof HBOL to 3 grams of ATX. The diffusion capacity coupled with theobservations about VO₂ and VCO₂ indicate that more oxygen is beingdelivered to the tissue level per gram of HBOL than ATX. It is commonlyheld that the diffusion capacity varies directly with the hemoglobinlevel, however, there is a suggestion that 1 gram of plasma hemoglobinmay increase diffusion capacity as much as 3 gms of RBC hemoglobin.

Laboratory studies were notable for small, but ransient increases inALT, AST, 5'-nucleotidase, lipase and creatine kinase during the HBOLperiod. There were no abnormal urinary finding.

Hematological studies were consistent with those in Example 5.

The observed medical events were consistent with expected findingsrelated to the phlebotomy (e.g., vasovagal episode), multiple pulmonaryfunction tests (eructation or abdominal "gas"), arterial line insertion(e.g., pain or tingling over the insertion site) or numerous everydaycomplaints that one might observe in normal subjects over the course ofa month (e.g., headache, upper respiratory tract infection or cold). Theone subject (Subject #105) that had abdominal "gas" and pressure in themid-epigastrium, but without dysphagia is suggestive of othergastrointestinal complaints that have been observed in previous HBOLstudies. L-arginine was used as a therapeutic measure based on theconcept that hemoglobin can interfere with endogenous nitric oxidefunction (nitric oxide is integral in the relaxation of gastrointestinalsmooth muscle, especially in the esophagus and intestines). L-arginineis the substrate upon which nitric oxide synthase produces nitric oxide.Theoretically, if one has a reduction in nitric oxide from thehemoglobin (perhaps binding of heme to nitric oxide), thenadministration of L-arginine might be of benefit. Apparently the subjectdid get marked by transient relief from his symptoms with the L-argininefor about two hours. This is not an unexpected finding because theplasma half-life of L-arginine is about an hour. Unfortunately, some ofthe side effects (nausea and vomiting) occurred and the infusion wasstopped. We elected to give him a two doses of an anticholinergic,antispasmodic drug, hyoscyamine. This apparently continued to reduce thesymptoms of abdominal "gas" and pressure. The subject had no furthercomplaints or sequelae.

In summary, HBOL was associated with improved oxygen delivery andutilization during exercise and at rest. HBOL produced a similarspectrum of hemodynamic, safety laboratory results, pharmacokinetics andmedical events to what has previously been observed. Intervention withL-arginine may produce a reversal of gastrointestinal symptoms, but itsuse was limited by nausea and vomiting. However, the use ofanticholinergic therapy might be of value in the treatment for thegastrointestinal symptoms that are encountered.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all other suchequivalents are intended to be encompassed by the following claims.

The invention claimed is:
 1. A method for producing a purifiedhemoglobin product, comprising the steps of:a) loading a hemoglobinsolution onto an anion exchange chromatography column; and b) injectingat least one tris(hydroxymethyl) aminomethane acetate buffer solutioninto the column, said buffer solution having a pH lower than that of thecolumn, whereby a purified hemoglobin product elutes from the column. 2.The method of claim 1, wherein at least two tris(hydroxymethyl)aminomethane acetate buffer solutions are injected sequentially into thecolumn, each said buffer solution having a distinct pH, whereby thecolumn is subjected to a stepped pH gradient.
 3. The method of claim 2,wherein the column is equilibrated with at least one of saidtris(hydroxymethyl) aminomethane acetate buffer solutions prior toelution of the hemoglobin product.
 4. The method of claim 3, wherein thecolumn is equilibrated at a pH in a range of between about 8.2 and about8.6.
 5. The method of claim 4, wherein the column initially isequilibrated at a pH above about 8.7 prior to injecting said buffersolutions.
 6. The method of claim 5, wherein tris(hydroxymethyl)aminomethane acetate buffer is employed to initially equilibrate saidcolumn.
 7. The method of claim 6, wherein the pH of initialequilibration is in a range of between about 8.7 and about 10.0.
 8. Themethod of claim 7, wherein the pH of initial equilibration is in a rangeof between about 8.7 and about 9.3.
 9. The method of claim 8, whereinthe pH of initial equilibration is in a range of between about 8.9 andabout 9.1.
 10. The method of claim 4, wherein at least about elevencolumn void volumes of the buffer solution are injected into the columnduring said equilibration of the column.
 11. The method of claim 10,wherein said equilibration is at a pH in a range of between about 8.2and about 8.4.
 12. The method of claim 11, wherein the hemoglobinproduct is eluted with a buffer at a pH in a range of between about 6.5and about 7.5.
 13. A method for producing a purified hemoglobin product,comprising the steps of:a) loading a hemoglobin solution onto an anionexchange chromatography column, said loaded column initially beingequilibrated to a pH greater than about 8.7; and b) injecting at leastone tris(hydroxymethyl) aminomethane acetate buffer solution into thecolumn, said tris(hydroxymethyl) aminomethane acetate buffer solutionhaving a pH lower than about 8.2, whereby a purified hemoglobin productelutes from the column.
 14. The method of claim 13, wherein said initialequilibration is at a pH in a range of between about 8.7 and about 10.0.15. The method of claim 14, wherein said initial equilibration is at apH in a range of between about 8.7 and about 9.3.
 16. The method ofclaim 15, wherein said initial equilibration is at a pH in a range ofbetween about 8.9 and about 9.1.
 17. The method of claim 16, wherein thecolumn is equilibrated initially with tris(hydroxymethyl) aminomethaneacetate.
 18. The method of claim 13, further including the step ofinjecting at least one tris(hydroxymethyl) aminomethane acetate buffersolution having a pH below about 8.6 into the column, saidtris(hydroxymethyl) aminomethane acetate intermediate buffer solutionhaving a pH between that at which the hemoglobin solution is loaded intothe column and a final pH, at which purified hemoglobin elutes from thecolumn, whereby the column is subjected to a stepped pH gradient. 19.The method of claim 18, wherein the column is equilibrated with at leastone said tris(hydroxymethyl) aminomethane acetate buffer solution priorto elution of the hemoglobin product.
 20. The method of claim 19,wherein the column is equilibrated with said intermediatetris(hydroxymethyl) aminomethane acetate buffer solution at a pH in arange of between about 8.2 and about 8.6.
 21. The method of claim 20,wherein at least about eleven column void volumes of saidtris(hydroxymethyl) aminomethane acetate intermediate buffer solutionare injected into the column during said equilibration of the column.22. The method of claim 21, wherein said equilibration with saidtris(hydroxymethyl) aminomethane acetate buffer solution is at a pH in arange of between about 8.2 and about 8.4.
 23. The method of claim 22,wherein the hemoglobin product is eluted at a pH in a range of betweenabout 6.5 and about 7.5.
 24. A method for producing a purifiedhemoglobin product, comprising the steps of:a) loading a hemoglobinsolution onto an anion exchange chromatography column; b) injecting tothe column at least eleven column void volumes of an equilibratingtris(hydroxymethyl) aminomethane acetate buffer solution having a pH ina range of between about 8.2 and about 8.6; and c) injecting into thecolumn a tris(hydroxymethyl) aminomethane acetate buffer solution havinga pH lower than that of the equilibrating buffer solution, whereby apurified hemoglobin product elutes from the column.
 25. The method ofclaim 24, wherein the equilibrating buffer solution has a pH of about8.3.
 26. The method of claim 24, wherein at least twotris(hydroxymethyl) aminomethane acetate buffer solutions are injectedsequentially into the column, each said buffer solution having adistinct pH, whereby the column is subjected to a stepped pH gradient.27. The method of claim 26, wherein the column initially is equilibratedat a pH above about 8.7 prior to injecting said buffer solutions. 28.The method of claim 27, wherein tris(hydroxymethyl) aminomethane acetatebuffer is employed to initially equilibrate said column.
 29. The methodof claim 28, wherein the pH of initial equilibration is in a range ofbetween about 8.7 and about 10.0.
 30. The method of claim 29, whereinthe pH of initial equilibration is in a range of between about 8.7 andabout 9.3.
 31. The method of claim 30, wherein the pH of initialequilibration is in a range of between about 8.9 and about 9.1.
 32. Themethod of claim 31, wherein the hemoglobin product is eluted at a pH ina range of between about 6.5 and about 7.5.
 33. A method for producing apurified hemoglobin product, comprising the steps of:a) loading ahemoglobin solution onto an anion exchange chromatography column, saidloaded column initially being equilibrated to a pH greater than about8.7; b) injecting into the column at least eleven column void volumes ofan equilibrating buffer solution of tris(hydroxymethane) aminomethaneacetate having a pH in a range of between about 8.2 and about 8.6; andc) injecting at least one tris(hydroxymethane) aminomethane acetatebuffer solution into the column, said buffer solution having a pH lowerthan about 8.2, whereby a purified hemoglobin product elutes from thecolumn.
 34. The method of claim 33, wherein tris(hydroxymethyl)aminomethane acetate buffer is employed to initially equilibrate saidcolumn.
 35. The method of claim 34, wherein the pH of initialequilibration is in a range of between about 8.7 and about 10.0.
 36. Themethod of claim 35, wherein the pH of initial equilibration is in arange of between about 8.7 and about 9.3.
 37. The method of claim 36,wherein the pH of initial equilibration is in a range of between about8.9 and about 9.1.
 38. The method of claim 37, wherein saidequilibrating buffer solution has a pH in a range of between about 8.2and about 8.4.
 39. The method of claim 38, wherein the hemoglobinproduct is eluted with a buffer at a pH in a range of between about 6.5and about 7.5.
 40. The method of claim 33, wherein the chromatographycolumns are packed with an anion exchange medium.
 41. The method ofclaim 40, wherein the anion exchange medium is selected from the groupconsisting of an amine- or ammonium-containing silica gel, alumina gel,titania gel, cross-linked dextran, agarose, a polyacrylamide, apolyhydroxyethyl-methacrylate or styrene divinylbenzene.
 42. The methodof claim 41, wherein the anion exchange medium is an amine or ammoniumcontaining silica gel.
 43. The method of claim 33, wherein the purifiedhemoglobin product includes less than 0.5 endotoxin units permilliliter, less than 3.3 nmoles/ml phospholipids and essentially nonon-hemoglobin proteins.